Methods for making and using sinoatrial node-like pacemaker cardiomyocytes and ventricular-like cardiomyocytes

ABSTRACT

Provided are methods for producing compositions comprising a population of cardiomyocytes enriched for or substantially devoid of sinoatrial node-like pacemaker cardiomyocytes (SANLCM) from human pluripotent stem cells (hPSCs), and methods of use thereof.

RELATED APPLICATIONS

This is a Patent Cooperation Treaty Application which claims the benefitof 35 U.S.C. § 119 based on the priority of U.S. Provisional PatentApplication No. 62/117,107, filed Feb. 17, 2015 which is incorporatedherein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing “P47907PC00 SequenceListing_ST25.txt” (5,578 bytes), submitted via EFS-WEB and created onFeb. 17, 2016, is herein incorporated by reference.

FIELD

The disclosure provides methods for producing and compositionscomprising sinoatrial node-like pacemaker cardiomyocytes (SANLCM) andventricular-like cardiomyocytes (VLCM) from human pluripotent stem cells(hPSC).

BACKGROUND

The sinoatrial node (SAN) is the primary pacemaker of the heart andfunctions to establish the heart rate throughout life^(1,2). Failure ofSAN function due to congenital disease or aging results in bradycardia,which eventually leads to circulatory collapse. The standard treatmentfor SAN failure patients is implantation of an electronic pacemaker thathas disadvantages including lack of hormonal responsiveness and risk ofinfections due to electronic leads^(3,4). Biological pacemakers derivedfrom human pluripotent stem cells (hPSCs) could represent a promisingalternative since advances in the stem cell field now allow efficientproduction of hPSC-derived cardiomyocytes (90%)⁵⁻⁷. The cardiomyocytepopulation consists of a mixture of ventricular, atrial and pacemakercells.^(8,9) However, there are currently only limited strategies tospecifically generate and isolate each of these cardiomyocyte subtypes.

In Zhu et al.²³, the progenitor cells having nodal phenotype producedfrom hPSCs were increased by inhibition of NRG-1β/ErbB signaling.Pacemaker cells were selected from hPSC-derived cardiomyocytepopulations using a GATA-6 promoter/enhancer eGFP reporter. The type ofnodal cells was not specified. Secondary atrioventricular (AVN) cellscan be identified with a GATA-6 reporter in the mouse heart, as shown inDavis et al.²⁴

In Kehat et al.³⁰, cardiomyocyte cell grafts were generated from hPSCsin vitro using an embryoid body differentiating system and weretransplanted into hearts of swine with atrioventricular block withoutselection for pacemaker cells. Only in 6 out of 13 animals a stableectopic rhythm activated by the human transplant could be seen.

In Ionta et al.³¹, it was reported that overexpression of SHOX2 bytransduction with an adenoviral vector expressing human SHOX2 duringmouse ESC differentiation upregulated the pacemaker gene program,resulting in enhanced automaticity in vitro and induced biologicalpacing upon transplantation in a rat.

SUMMARY

An aspect includes a method of producing a population of cardiomyocytesfrom human pluripotent stem cells (hPSCs), the steps comprising:

-   -   a. incubating the hPSCs in an embryoid body medium comprising a        BMP component, optionally BMP4, optionally further comprising a        Rho-associated protein kinase (ROCK) inhibitor, for a period of        time to generate embryoid bodies;    -   b. incubating the embryoid bodies in a mesoderm induction medium        comprising a BMP component, optionally BMP4, and an activin        component, optionally Activin A, and optionally a FGF component,        optionally bFGF, for a period of time to generate cardiovascular        mesoderm cells;    -   c. incubating the cardiovascular mesoderm cells:        -   i.            -   1. in a cardiac induction medium comprising a BMP                component, optionally BMP4, above a selected amount, and                retinoic acid (RA), and optionally one or more of a FGF                inhibitor, a WNT inhibitor, optionally IWP2, VEGF and an                activin/nodal inhibitor, optionally SB-431542; for a                period of time to generate cardiovascular progenitor                cells that express TBX18 wherein the cardiovascular                mesoderm cells are preferably incubated with the FGF                inhibitor and which FGF inhibitor is provided for all or                part of a cardiac induction phase; and            -   2. incubating the cardiovascular progenitor cells in a                basic medium comprising VEGF for a period of time to                generate a population of cardiomyocytes enriched for                SANLCMs; or        -   ii.            -   1. in a cardiac induction medium comprising one or more                of a WNT inhibitor, optionally IWP2, and VEGF; and                optionally a FGF component and/or an activin/nodal                inhibitor, optionally SB-431542; for a period of time to                generate cardiovascular progenitor cells that express                NKX2-5 and            -   2. incubating the cardiovascular progenitor cells in a                basic medium comprising VEGF for a period of time to                generate a population of cardiomyocytes that are                enriched for NKX2-5^(pos) cTNT^(pos) cells and                substantially devoid of SANLCMs; and    -   d. optionally isolating the population of cardiomyocytes using a        cardiomyocyte-specific surface marker, optionally wherein the        marker is signal-regulatory protein alpha (SIRPA), and thymocyte        differentiation antigen 1 (THY-1/CD90) optionally wherein the        isolated population is SIRPA^(pos) CD90^(neg).

In an embodiment, the mesoderm induction medium comprises BMP4 at aconcentration of about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4ng/mL, about 5 ng/mL, about 8 ng/mL, about 10 ng/mL, or up to or about20 ng/ml optionally about 3 ng/mL, and/or Activin A at a concentrationof up to or about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 5 ng/mL,about 8 ng/mL, about 10 ng/mL, or up to about 20 ng/mL optionally about2 ng/mL, preferably wherein the mesoderm induction medium comprises BMP4at a concentration of about 3 ng/mL and Activin A at a concentration ofabout 2 ng/mL.

In another embodiment, the cardiovascular mesoderm cells are incubatedwith BMP4 for about 1 day to about 4 days, optionally 2 days or 3 days,at a concentration of about 0.5 ng/mL, about 1.0 ng/mL, about 1.5 ng/mL,about 2.0 ng/mL, about 2.5 ng/mL, about 3.0 ng/mL, about 5.0 ng/mL,about 10.0 ng/mL, about 20.0 ng/mL, or about 40.0 ng/mL, or about 80.0ng/mL, optionally about 2.5 ng/mL, and with RA for about 1 day to about2 days, optionally 1 day, at a concentration of about 20 ng/mL, about 50ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 300ng/mL, about 400 ng/mL, about 500 ng/mL, or about 1000 ng/mL, optionallyabout 150 ng/mL. An aspect includes a method of producing a populationof cardiomyocytes enriched for sinoatrial node-like pacemakercardiomyocytes (SANLCM) from human pluripotent stem cells (hPSCs), thesteps comprising:

-   -   a. incubating the hPSCs in an embryoid body medium comprising a        BMP component, optionally BMP4, optionally further comprising a        Rho-associated protein kinase (ROCK) inhibitor, for a period of        time to generate embryoid bodies;    -   b. incubating the embryoid bodies in a mesoderm induction medium        comprising a BMP component, optionally BMP4, and an activin        component, optionally Activin A and optionally a FGF component,        optionally bFGF, for a period of time to generate cardiovascular        mesoderm cells;    -   c.        -   i. incubating the cardiovascular mesoderm cells in a cardiac            induction medium comprising a BMP component, optionally            BMP4, above a selected amount, and retinoic acid (RA), and            optionally one or more of a FGF inhibitor, a WNT inhibitor,            optionally IWP2, VEGF and an activin/nodal inhibitor,            optionally SB-431542; for a period of time to generate            cardiovascular progenitor cells that express TBX18; wherein            the cardiovascular mesoderm cells are preferably incubated            with the FGF inhibitor and which FGF inhibitor is provided            for all or part of the cardiac induction phase; and        -   ii. incubating the cardiovascular progenitor cells in a            basic medium comprising VEGF for a period of time to            generate a population of cardiomyocytes enriched for            SANLCMs; and    -   d. optionally isolating the population of cardiomyocytes        enriched for SANLCMs using a cardiomyocyte-specific surface        marker, optionally wherein the marker is signal-regulatory        protein alpha (SIRPA), and thymocyte differentiation antigen 1        (THY-1/CD90) optionally wherein the isolated population is        SIRPA^(pos) CD90^(neg).

In embodiments for producing a population of cardiomyocytes enriched forSANLCM, the cardiovascular mesoderm cells are preferably incubated in acardiac induction medium comprising BMP4 above a selected amount, RA,and a FGF inhibitor.

In an embodiment, the FGF inhibitor is selected from PD 173074 (Torcis),SU 5402 (Torcis), and any other FGF receptor inhibitor or FGF signalinginhibitor.

In an embodiment, the cardiovascular mesoderm cells are incubated withthe FGF inhibitor for at about 2 to about 7 days, optionally about 2days, about 3 days, about 4 days or about 5 days.

An aspect includes a method of producing a population of cardiomyocytessubstantially devoid of SANLCM from human pluripotent stem cells(hPSCs), the steps comprising:

-   -   a. incubating the hPSCs in an embryoid body medium comprising a        BMP component, optionally BMP4, optionally further comprising a        Rho-associated protein kinase (ROCK) inhibitor, for a period of        time to generate embryoid bodies;    -   b. incubating the embryoid bodies in a mesoderm induction medium        comprising a BMP component, optionally BMP4, and an activin        component, optionally Activin A and optionally a FGF component,        optionally bFGF, for a period of time to generate cardiovascular        mesoderm cells;    -   c.        -   i. incubating the cardiovascular mesoderm cells in a cardiac            induction medium comprising one or more of a WNT inhibitor,            optionally IWP2, and VEGF; and optionally a FGF component            and/or an activin/nodal inhibitor, optionally SB-431542; for            a period of time to generate cardiovascular progenitor cells            that express NKX2-5; and        -   ii. incubating the cardiovascular progenitor cells in a            basic medium comprising VEGF for a period of time to            generate a population of cardiomyocytes that are enriched            for NKX2-5^(pos) cTNT^(pos) cells and substantially devoid            of SANLCMs; and    -   d. optionally isolating the population of cardiomyocytes using a        cardiomyocyte-specific surface marker, optionally wherein the        marker is signal-regulatory protein alpha (SIRPA) and thymocyte        differentiation antigen 1 (THY-1/CD90) optionally wherein the        isolated population is SIRPA^(pos) CD90^(neg).

In embodiments directed to obtaining a population of cardiomyocytessubstantially devoid of SANLCM, the cardiovascular mesoderm cells arepreferably incubated with FGF component, optionally bFGF.

In an embodiment, the cardiovascular mesoderm cells are incubated withthe FGF component for at about 2 to about 7 days, optionally about 2days, about 3 days, about 4 days or about 5 days.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1. Enrichment for sinoatrial node pacemaker-like cardiomyocytes bysorting for NKX2-5 negative, SIRPA positive cells. A, Scheme of thedevelopmentally staged protocol used for hESC differentiation intocardiomyocytes. B, Q-PCR analysis of pacemaker markers at different timepoints during differentiation. C, Representative FACS plots forSIRPA/cTNT and NKX2-5 expression during differentiation using theNKX2-5:GFP hESC line. D, Representative FACS plots of sorting strategyfor NKX2-5⁺SIRPA⁺CD90⁻ and NKX2-5⁻SIRPA⁺CD90⁻ populations at day 20 ofdifferentiation. E, Brightfield and GFP channel overlay image ofNKX2-5⁺SIRPA⁺ and NKX2-5⁻SIRPA⁺ sorted populations and quantification ofbeating rates (n=30). Scale bars represent 200 μm. F-I, Q-PCR analysisof: pan-cardiomyocyte and ventricular marker (f), SAN pacemaker marker(G), AVN pacemaker marker (H) and cardiac ion channels (i), inNKX2-5⁺SIRPA⁺ and NKX2-5⁻SIRPA⁺ sorted cells. Fetal tissue was used asexpression reference. Expression relative to housekeeping gene TBP,normalized to NKX2-5⁺SIRPA⁺ for hESC-derived cell populations and F-Vfor fetal tissue samples. T-test: *P<0.05, **P<0.01 vs NKX2-5⁺SIRPA⁺cells, ^(#)P<0.05, ^(##)P<0.01 vs F-V or indicated sample (n=5). Errorbars show s.e.m. J,K, Immunostaining for ventricular contractileapparatus marker MLC2V (j) and pacemaker transcription factor SHOX2 (K)in NKX2-5⁺SIRPA⁺ and NKX2-5⁻SIRPA⁺ sorted cells from day 20 cultures.Cells were counterstained with cTNT to mark cardiomyocytes and DAPI tomark cell nuclei. Scale bars represent 100 μm. BPM, beats per minute; d,day; PS, primitive streak; F-V, fetal ventricle; F-SAN, fetal sinoatrialnode; F-AVN, fetal atrioventricular node.

FIG. 2. BMP4 signaling specifies the SANLCM pacemaker population. A,Scheme of modified differentiation protocol with dissociation of EBs atthe mesoderm stage (day 3) to efficiently deliver signaling molecules.B-D, Summary of day 20 FACS results for NKX2-5 and cTNT after BMP4titration from day 3-5 in cultures that were induced for mesodermformation with: 10 ng/ml BMP4, 6 ng/ml ACTA (B); 5 ng/ml BMP4, 4 ng/mlACTA (C); 3 ng/ml BMP4, 2 ng/ml ACTA (D). T-test: *P<0.05, **P<0.01 vsNKX2-5⁻ CTNT⁺ cells at endogenous (E) BMP4 levels (n=4). E,Representative FACS plots at day 20 of differentiation, showing theincrease of the NKX2-5⁻cTNT⁺/NKX2-5⁻SIRPA⁺ SANLCM population in culturestreated with 2.5 ng/ml of BMP4 from day 3-5 of differentiation. F,Summary of FACS results analyzing the time window of BMP4 treatment thatis efficient to increase the SANLCM population. T-test: *P<0.05,**P<0.01 vs untreated control (n=3). G, Q-PCR analysis of TBX18expression in untreated control vs BMP4 (day 3-5, 2.5 ng/ml) treatedcultures from day 4 to day 12 of differentiation (n=3). H,Quantification of TBX18⁺ cells from immunostaining (n=3). I,Immunostaining for TBX18 in control and BMP4 treated cultures at day 6of differentiation. DAPI was used to counterstain cell nuclei. Scalebars represent 100 μm. Error bars show s.e.m. T-test: *P<0.05, **P<0.01.d, day; Dorso, dorsomorphin; E, endogenous/untreated.

FIG. 3. Retinoic acid signaling enhances SAN pacemaker phenotype. A,Summary of FACS results analyzing the effect of retinoic acid (RA)treatment at different time points during differentiation on the size ofthe NKX2-5⁻cTNT⁺ SANLCM population (n=4). B, Q-PCR analysis of TBX5 andSHOX2 at day 20 of differentiation in NKX2-5⁻SIRPA⁺ sorted SANLCM fromcultures that were treated with RA at indicated time points. Expressionrelative to the housekeeping gene TBP. T-test: *P<0.05, **P<0.01 vsuntreated control (n=4). C, Summary of FACS results for the effect ofBMP4 (day 3-5), RA (day 3) and combined RA/BMP4 treatment on the size ofthe SANLCM population. T-test: **P<0.01 D-F, Q-PCR analysis of theeffect of BMP4, RA and combined RA/BMP4 treatment on the expression of:SAN pacemaker marker (D), ventricular marker and cardiac ion channels(E) and AVN pacemaker marker (F) in NKX2-5⁻SIRPA⁺ sorted SANLCM from day20 cultures. Expression relative to the housekeeping gene TBP. T-test:*P<0.05, **P<0.01 vs untreated control (n=5). G, Effect of BMP4, RA andcombined RA/BMP4 treatment on the beating rates of SANLCM at day 20 ofdifferentiation. T-test: *P<0.05, **P<0.01 vs untreated control,^(#)P<0.05, vs indicated sample (n=10). H, Summary Scheme of suggestedmodel of SANLCM lineage specification from hPSCs by BMP4 and RAsignaling. Error bars show s.e.m. d, day; RA, retinoic acid.

FIG. 4. SANLCM have functional pacemaker character. A,B, Original tracesof spontaneous action potentials recorded for NKX2-5⁺SIRPA⁺ sorted VLCM(A) and NKX2-5⁻ SIRPA⁺ sorted SANLCM (B). C, Histogram plot showing thedistribution of the maximum upstroke velocities (dV/dt_(max)) recordedin SANLCM and VLCM. D, Original traces of pacemaker funny current(I_(f)) at different membrane potentials, recorded in SANLCM and averageof maximum I_(f) current density at −120 mV in VLCM and SANLCM (n=12).E, Original traces of sodium current (I_(Na)) at different membranepotentials, recorded in VLCM and average of maximum I_(Na) currentdensity at −20 mV in VLCM and SANLCM (n=11). T-test: **P<0.01 vs VLCM.F, Scheme of experimental setup used for in-vitro pacing experiments ofVLCM monolayers cultured on multi-electrode arrays. G-I, VLCM monolayerbefore addition of SANLCM aggregate. Low magnification bright fieldpicture of VLCM monolayer (G). Representative greyscale-map ofelectrical signal propagation in the depicted monolayer. Thegreyscale-map represents a snapshot of the electrical propagation in themonolayer and shows that the electrical signal is initiated at thebottom left corner (white) and is propagated to the upper right corner(black) (H). Beating rate of the monolayer in beats per minute (bpm)presented as original field potentials recorded at electrode 65 (I).J-L, VLCM monolayer after addition of Tetramethylrhodamine methyl ester(TMRM) labeled SANLCM aggregate. Low magnification bright field andTMRM, GFP channel pictures. The SANLCM aggregate is located on top ofelectrode 65. Insets: 2.5-fold magnification (J). Representativegreyscale-map of electrical propagation in the monolayer after placingthe SANLCM aggregate. Original field potentials recorded at electrodes65, 46, and 28 highlight that the electrical signal is initiated by theSANLCM aggregate (electrode 65) (K). Beating rate of the culture inbeats per minute (bpm) depicted below as original field potentialsrecorded at electrode 65 and electrode 28 (L). Scale bars represent 250μm. Error bars show s.e.m.

FIG. 5. A, Representative FACS plot for PDGFRα⁺, KDR⁺ cardiac mesodermat day 4 of differentiation and resulting cTNT population at day 20 ofdifferentiation. B, Q-RT-PCR analysis of mesoderm and cardiac markers atdifferent time points during differentiation. C, Representative FACSplot of cTNT analysis in the NKX2-5⁺SIRPA⁺ and NKX2-5⁻ SIRPA⁺populations after sort. D, Representative FACS plot of sorting strategyfor NKX2-5⁺cTNT⁺ and NKX2-5⁻ cTNT⁺ cells from PFA fixed day 20 cultures.E-H, Q-PCR analysis of: sorting markers (E), AVN pacemaker marker (F),SAN pacemaker marker (G), ventricular marker and cardiac ion-channels(H), in NKX2-5⁺cTNT⁺ and NKX2-5⁻ cTNT⁺ sorted cells. Expression relativeto housekeeping gene TBP. T-test: *P<0.05, **P<0.01 vs NKX2-5⁺cTNT⁺cells (n=3). Error bars show s.e.m.

FIG. 6. A, Immunostaining for pacemaker transcription factor TBX3 inNKX2-5⁺SIRPA⁺ and NKX2-5⁻ SIRPA⁺ sorted cells from day 20 cultures.Cells were counterstained with cTNT to mark cardiomyocytes and DAPI tomark cell nuclei. B, Immunostaining for cTNT in NKX2-5⁺SIRPA⁺ andNKX2-5⁻ SIRPA⁺ sorted cells at 30 days post-sorting. The NKX2-5:GFPtransgene expression was visualized to proof that NKX2-5⁻ sortedcardiomyocytes stay NKX2-5 negative for up to 30 days after the sort.Scale bars represent 100 μm. C, Representative FACS plot ofNKX2-5⁺SIRPA⁺ and NKX2-5⁻ SIRPA⁺ sorted cells for NKX2-5:GFP and cTNT at30 days post-sorting.

FIG. 7. A, Analysis of total cTNT and NKX2-5⁺/NKX2-5⁻ proportion ofcTNT⁺ cells after induction of mesoderm with indicated amounts of BMPand ACTA at day 1 of differentiation. B, Total number of cells at day 20of differentiation after treatment with different concentrations ofeither Dorsomorphin (Dorso) or BMP4 (n=4). Error bars show s.e.m.

FIG. 8. A, Summary of day 20 FACS results for NKX2-5 and cTNT afterretinoic acid (RA) (day 3), BMP4 (day 3-5) and combined RA+BMP4treatment. T-test: **P<0.01 vs NKX2-5⁻ cTNT⁺ cells in untreated controlcondition (n=4). B,C, Representative FACS plots for NKX2-5 and cTNTstaining in day 20 cultures of the H7 hESC line (B) and the MSC-iPS1iPSC line (c) differentiated with 500 nM RA at day 3 and the indicatedBMP4 concentrations from day 3-5. D, Representative FACS plot forsorting strategy of NKX2-5⁺cTNT⁺ and NKX2-5⁻cTNT⁺ cells. Due to the lackof a SANLCM specific surface marker MSC-iPS1 cultures were PFA fixed atday 20 and stained for NKX2-5 and cTNT. E-G, Q-PCR analysis of: sortingmarkers (E) AVN pacemaker marker (F) SAN pacemaker marker (G).Expression relative to the housekeeping gene TBP. T-test: *P<0.05,**P<0.01 vs NKX2-5⁺cTNT⁺ cells (n=4). Error bars show s.e.m.

FIG. 9. A, Current-voltage relationship for pacemaker funny currentdensity (I_(f)) in SANLCM and VLCM. Inset: voltage protocol, from aholding potential of −40 mV currents were elicited by 3 s voltage stepsof −10 mV down to −120 mV. Current densities were measured bysubtraction of the instantaneous current (t=0) from the current at theend of the 3 sec voltage step. B,C, Original pacemaker funny currenttraces at different membrane potentials in Tyrode's solution (B) andblocked by the application of 2 mM Cesium (Cs⁺) (C). Inset: voltageprotocol, from a holding potential of −40 mV currents were elicited by 3s voltage steps of −10 mV down to −120 mV. D, Current-voltagerelationship for sodium current density (I_(Na)) in SANLCM and VLCM.Inset: voltage protocol, from a holding potential of −100 mV currentswere elicited by 80 ms voltage steps of 5 mV up to +40 mV. Currentdensities were measured as peak inward currents. E,F, Analysis of barium(Ba²⁺)-sensitive inward rectifier potassium current density (I_(K1)):Original traces of the barium sensitive inward rectifier potassiumcurrent component at different membrane potentials (E). Current-voltagerelationship for barium-sensitive current density and average of maximumI_(K1) current density at −120 mV in VLCM and SANLCM. (F). Inset:voltage protocol, from a holding potential of −40 mV currents wereelicited by 3 s voltage steps of −10 mV down to −120 mV. T-test: *P<0.05vs maximum current density in VLCM. Error bars show s.e.m.

FIG. 10. A-C, VLCM Monolayer before addition of VLCM control aggregate.Low magnification bright field picture of VLCM Monolayer (A).Representative greyscale-map of electrical signal propagation in thedepicted monolayer. The greyscale-map represents a snapshot of theelectrical propagation in the monolayer and shows that the electricalsignal is initiated at the bottom right corner (white) and is propagatedto the upper left corner (black) (B). Beating rate of the monolayer inbeats per minute (bpm) presented as original field potentials recordedat electrode 65 (C). D-F, VLCM Monolayer after addition ofTetramethylrhodamine methyl ester (TMRM) labeled VLCM control Aggregate.Low magnification bright field and TMRM, GFP channel pictures. VLCMcontrol Aggregate is located on top of electrode 65. Insets: 2.5-foldmagnification. Note, the VLCM monolayer and the VLCM control aggregateare NKX2-5:GFP⁺ (D). Representative greyscale-map of electricalpropagation in the monolayer after placing the VLCM control aggregate.Original field potentials recorded at electrodes 65, 46, and 28highlight that the electrical signal is not initiated by the controlVLCM aggregate (electrode 65) (E). Beating rate of the culture in beatsper minute (bpm) depicted below as original field potentials recorded atelectrode 65 and electrode 28 (F). Scale bars represent 250 μm.

FIG. 11. SANLCM can act as biological pacemaker in-vivo. A, B, Exampleof rat heart with VLCM transplant (1-2×10⁶ cells) harvested 14 dayspost-transplantation. Original ECG traces before and after theapplication of Methacholine+Lidocaine in the Langendorff isolated heartmodel (A). Picture of rat heart with indicated transplantation site(ellipse) and whole heart optical mapping of the electric activitycorresponding to the ECG trace (B). C,D, Example of rat heart withSANLCM transplant (1-2×10⁶ cells) harvested 14 dayspost-transplantation. Original ECG traces before and after theapplication of Methacholine+Lidocaine in the Langendorff isolated heartmodel (C). Picture of rat heart with indicated transplantation site(ellipse) and whole heart optical mapping of the electric activitycorresponding to the ECG trace (D). Of note, the initiation site of theectopic beats correlates with the transplantation site. E,F,Immunostaining of cryosections of rat hearts with VLCM (E) and SANLCM(F) transplant. A human specific cTnT (hcTnT) antibody was used toidentify the human transplant. Sections were counterstained with cTNT tomark rat and human cardiomyocytes. Sections were stained for MLC2V todistinguish the VLCM (MLC2V⁺) and the SANLP (MLC2V⁻) transplant. DAPIwas used to mark cell nuclei. Dashed line indicates the humantransplant. Scale bars represent 200 μm. TP, transplant.

FIG. 12. SANLCM can be isolated from hPSC lines without transgenicNKX2-5:GFP reporter. (A) Flow cytometric analyses of day 20 cultures forthe proportion of NKX2-5⁺cTNT⁺ and NKX2-5⁻cTNT⁺ cells after treatmentwith BMP (2.5 ng/ml) and RA (0.5 μM) from day 3-6 and either bFGF (20ng/ml) or FGFi (480 nM) from day 4-6. Error bars represent s.e.m.One-way ANOVA followed by Bonferroni's post hoc test: **P<0.01 vsNKX2-5⁻cTNT⁺ cells at endogenous (E) bFGF levels, ^(##)P<0.01 vsNKX2-5⁺cTNT⁺ cells at endogenous (E) bFGF levels (n=4). (B)Representative flow cytometric analyses showing the proportion ofNKX2-5⁻cTNT⁺ SANLCMs and NKX2-5⁺cTNT⁺ cells in day 20 populations thatwere treated with BMP(2.5 ng/ml) and RA(0.5 μM) from day 3-6 and eitherbFGF (20 ng/ml) or no additional bFGF (endogenous) or FGFi (480 nM) fromday 4-6. (C) Flow cytometric analyses of day 20 cultures for theproportion of NKX2-5⁺cTNT⁺ and NKX2-5⁻cTNT⁺ cells after treatment withBMP (2.5 ng/ml) and RA (0.5 μM) from day 3-6 and indicated amounts ofFGFi from day 4-6. Error bars represent s.e.m. (n=2). (D) Flowcytometric analyses of day 20 cultures for the proportion ofNKX2-5⁺cTNT⁺ and NKX2-5⁻cTNT⁺ cells after treatment with BMP (2.5 ng/ml)and RA (0.5 μM) from day 3-5 and FGFi (480 nM) at indicated time points.Error bars represent s.e.m. One-way ANOVA followed by Bonferroni's posthoc test: **P<0.01 vs NKX2-5⁻cTNT⁺ cells in untreated Control,^(##)P<0.01 vs NKX2-5⁺cTNT⁺ cells in untreated Control (n=3). (E)Representative flow cytometric analyses of live cultures showing theproportion of NKX2-5⁻SIRPA⁺ cells and CD90⁻SIRPA⁺ cells in day 20populations specified with BMP and RA at day 3 and FGFi at day 4. Toenrich for SANLCMs cultures were sorted based on SIRPA⁺CD90⁻ expression(highlighted quadrant) and checked for their post-sort purity byNKX2-5:GFP and cTNT flow cytometric analysis on fixed cells. (F)Representative flow cytometric analyses of HES2 hESC-line derived day 20cultures that were specified with BMP (2.5 ng/ml) and RA (0.5 μM) fromday 3-5 and either bFGF (20 ng/ml) or no additional bFGF (endogenous) orFGFi (240 nM) from day 3-5 showing the proportion of NKX2-5⁻cTNT⁺SANLCMs and NKX2-5⁺cTNT⁺ cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

I. Definitions

The term “sinoatrial node-like cardiomyocytes” or “SANLCM” as usedherein refers to a subtype of cardiomyocytes that express sinoatrialnodal (SAN) cell specific markers TBX18, TBX3, SHOX2 and ISL1 and whichhave pacemaker activity. Like SAN cells, SANLCMs express low levels ofNKX2-5 and have faster beating rates (e.g. beats per minute) than othercardiomyocyte subtypes such as ventricular-like cardiomyocytes.

The term “ventricular-like cardiomyocytes” or “VLCM” as used hereinrefers to a subtype of cardiomyocytes expressing ventricular specificmarkers MYL2 and IRX4, as well as elevated levels of NKX2-5.

The term “atrioventricular (AVN) cardiomyocytes” as used herein is asubtype of cardiomyocytes that express TBX2 and MSX2 and can act as asecondary pacemaker. AVN cardiomyocytes are distinguishable from SANLCMswhich do not express TBX2 and/or MSX2

The term “cardiomyocyte” as used herein is a cardiac lineage cell thatexpresses cTNT and SIRPA.

The term “NKX2-5” as used herein refers to the cardiac homeobox proteinNKX2-5 encoded in humans by the NKX2-5 gene. The gene is involved in thecardiac differentiation and is expressed in cardiomyocyte subtypes suchas ventricular cardiomyocytes. Expression of NKX2-5 can be measuredusing for example an antibody specific to NKX2-5 or for example by usinga NKX2-5 reporter construct.

The term “NKX2-5 negative cell” as used herein means a cell with nodetectable NKX2-5 protein expression and/or a cell with detectableexpression below a selected threshold when analyzed for example by flowcytometry. For example, NKX2-5 expression can be detected using areporter assay such as a GFP reporter assay described herein or using anintracellular antibody for NKX2-5 protein. When using an intracellularantibody, an aliquot of cells to be tested are fixed, and stained asdescribed in FIG. 12F and a method described in the Examples section.

The threshold for example can be based on a mixed population ofcardiomyocyte lineage cells prepared according to a method describedherein wherein the threshold is selected based on an expression levelthat separates the mixed population into two groups. The threshold canalso be based on comparison to a control. For example, the control whenstaining cells for NKX2-5 expression can be undifferentiated hPSCs or afibroblast cell line which are cells known to not express NKX2-5. Asused herein, “NKX2-5 negative cell” also means a cell comprising aNKX2-5 reporter, for example a NKX2-5:GFP reporter, with no detectableexpression of the fluorescent protein when analyzed, for example by flowcytometry or expression below a threshold or relative to a control.—Forexample, in the case of an NKX2-5 reporter line a good negative controlwould be to use cardiomyocytes that were differentiated from a hPSC linethat does not carry the reporter. In addition, an internal control canbe used. For example, cells can be co-stained with an antibody for cTNT.In such case cTNTneg non-cardiomyocytes will not express NKX2-5 and canbe used as an internal negative control.

The term “BMP component” as used herein means any molecule optionallyany BMP or growth and differentiation factor (GDF) that activates thereceptor for BMP4, including for example BMP4 and BMP2.

The term “BMP4” (for example Gene ID: 652) as used herein refers to BoneMorphogenetic Protein 4, for example human BMP4, as well as activeconjugates and fragments thereof, that can for example activate BMP4receptor signlaing.

The term “activin component” as used herein means one or morecomponents, or a composition comprising said component(s) that activatesnodal signal transduction, optionally which has Activin A activity suchas Activin A and/or nodal.

The term “activin” or “ActA” as used herein refers to “Activin A”, (e.g.Gene ID: 3624), for example human activinA, as well as active conjugatesand fragments thereof, that can activate nodal signal transduction.

The term “activin/nodal inhibitor” and/or “activin/nodal/TGF-βRinhibitor” as used herein means any molecule that inhibits signal of theactivin/nodal pathway and particularly any molecule that inhibitsreceptors ALK4, ALK7 and/or TGF-βRI, including but not limited toSB431542 (Sigma Aldrich) A83-01 (Tocris, 2929), D 4476, GW 788388, LY364947, RepSox, SB 505124, SB 525334 (Sigma Aldrich), and SD 208.

The term “wnt inhibitor” as used herein means any agent, including anycompound and/or protein that inhibits wnt signaling, including but notlimited to wnt antagonists that bind either to the Wnt ligand itself, orto Wnt receptors, such as Dickkopf (Dkk) proteins, Wnt InhibitoryFactor-1 (WIF-1), and secreted Frizzled-Related Proteins (sFRPs), aswell as wnt inverse agonists (e.g. an agent that binds to the samereceptor as an agonist but induces a pharmacological response oppositeto that of an agonist). Examples of Wnt inhibitors include XAV939, IWP2, an inhibitor of wnt processing, and iCRT14, which is a potentinhibitor of β-catenin-responsive transcription (CRT), both of which areavailable from Tocris Bioscience, as well as combinations thereof.

The term “FGF component” as used herein means a molecule such as acytokine, including for example FGF, or a small molecule, that activatesa FGF signalling pathway, e.g. binds and activates a FGF receptor. Theterm “FGF” as used herein refers to any fibroblast growth factor, forexample human FGF1 (Gene ID: 2246), FGF2 (also known as bFGF; Gene ID:2247), FGF3 (Gene ID: 2248), FGF4 (Gene ID: 2249), FGF5 (Gene ID: 2250),FGF6 (Gene ID: 2251), FGF7 (Gene ID: 2252), FGF8 (Gene ID: 2253), FGF9(Gene ID: 2254) and FGF10 (Gene ID: 2255) optionally including activeconjugates and fragments thereof, including naturally occuring activeconjugates and fragments. In certain embodiments, FGF is bFGF, FGF10,FGF4 and/or FGF2.

The term “retinoic acid” or “RA” includes vitamin A and metabolites ofvitamin A that mediate the function of vitamin A, and includes forexample all-trans RA (e.g. Sigma R2625), 9-cis RA (e.g. Sigma R4643),and retinol (e.g. Sigma R7632) as well as RA analogs (e.g. RARβagonists), such as AM580, a selective RARα agonist (Tocris 0760),AC55649, a selective RARβ agonist (Tocris 2436), and CD437, a selectiveRARγ agonist (Tocris 1549).

The term “FGF inhibitor” as used herein means any FGF receptor inhibitor(FGFR 1,2,3,4) or FGF signaling inhibitor (i.e. downstream p38 MAPKinhibitor), including but not limited to PD 173074 (Tocris) and SU 5402(Torcis) and p38 MAPK inhibitor SB203580 (Tocris).

The term “SIRPA” as used herein refers to the signal-regulatory proteinalpha (SIRPA) pan-cardiomyocyte cell surface marker, including forexample human SIRPA (e.g. GENE ID: 140885).

The term “embryoid body medium” as used herein is a culture medium thatsupports formation of aggregates (e.g. floating aggregates) fromembryonic/pluripotent stem cells and comprising a minimal media such asStemPro 34 (ThermoFisher), MesoFate™ (Stemgent), RPMI (ThermoFisher andother companies), HES-media (DMEM/F12 with KnockOut Serum Replacement,ThermoFisher and other companies) and for example a BMP component,optionally BMP4, and further optionally comprising a Rho-associatedprotein kinase (ROCK) inhibitor. An example of an embryoid body mediumis provided in Example 1.

The term “embryoid body aggregation phase” as used herein means the timeperiod non-aggregated hPSCs are cultured for example with an embryoidbody medium described herein and are treated with BMP component and aswell as optionally ROCK inhibitor and/or other components that result inembryoid bodies (i.e. aggregates of embryonic/pluripotent stem cellsthat can be differentiated). The component treatments can besimultaneous, overlapping or distinct. For example, a first componentcan be comprised in the medium and a second component can be added tothe medium during the embryoid body aggregation phase.

The term “mesoderm induction medium” as used herein is a culture mediumthat supports the formation of cardiovascular mesoderm cells andcomprises a minimal media such as StemPro 34 (ThermoFisher), MesoFate™(Stemgent), RPMI (ThermoFisher and other companies), and for example aBMP component, optionally BMP4, above a selected amount, and an activincomponent, optionally Activin A and optionally comprising a FGFcomponent, optionally bFGF. An example of a mesoderm induction medium isprovided in Example 1.

The term “mesoderm induction phase” as used herein means the time periodmesoderm cells are cultured with mesoderm induction medium and aretreated with BMP component and an activin component as well asoptionally FGF component and/or other components that result incardiovascular mesoderm cells. The component treatments can besimultaneous, overlapping or distinct. For example, a first componentcan be comprised in the medium and a second component can be added tothe medium during the mesoderm induction phase.

The term “cardiac induction medium” as used herein is a culture mediumthat supports cardiac progenitor cells such as StemPro-34 minimal mediacomprising for example a WNT inhibitor, optionally IWP2, VEGF and/or anoptionally activin/nodal inhibitor, optionally SB-431542. Depending onthe desired cell type, the cardiac induction medium may also comprise aBMP component, retinoic acid, an FGF inhibitor or a FGF component. Anexample of a cardiac induction medium is provided in Example 1. Anexample of a cardiac induction medium (also referred to as standardcardiac induction media) is StemPro-34 minimal media containing 0.5 μMIWP2, 10 ng/ml VEGF, and optionally 5.4 μM SB-431542. Other minimalmedia that can be used include MesoFate™ (Stemgent) and RPMI(ThermoFisher and other companies).

The term “cardiac induction phase” as used herein means the time periodcardiac progenitor cells are cultured with cardiac induction medium andare treated for example with BMP component and RA as well as optionallyan FGF inhibitor or FGF component and/or other components that result incardiovascular progenitor cells. The treatments can be simultaneous,overlapping or distinct. For example, a first component can be comprisedin the medium and a second component can be added to the medium duringthe cardiac induction phase.

The term “basic medium” as used here is a culture medium that supportsgrowth of cardiovascular progenitor cells and cardiomyocytes comprisinga minimal media such as StemPro 34 (ThermoFisher), MesoFate™ (Stemgent),RPMI (ThermoFisher and other companies), and for example VEGF. Anexample of a basic medium is provided in Example 1.

The term “basic phase” as used herein means the time periodcardiovascular progenitor cells are cultured with basic medium and aretreated with VEGF and/or other components that result in cardiomyocytes.The treatments can be simultaneous, overlapping or distinct.

The term “incubating” as used herein includes any in vitro method ofmaintaining and/or propagating a population of cells, includingmonolayer, bead, flask, or 3D cultures, optionally where ambientconditions are controlled as in an incubator and optionally involvingpassaging of cells. Steps that involve incubating the cells with more ormore components, the components can be added simultaneously, atdifferent times, for overlapping periods or for distinct periods. Afactor can be added to the medium after the cells have startedincubating in for example an induction medium or the factor can be addedto the medium before the medium is added to the cells. Further, cellsmay be washed between incubations, for example to reduce the level of acomponent from a previous incubation.

The term “culturing” as used herein means any in vitro method ofmaintaining and propagating a population of cells at least through onecell division, including monolayer, bead, flask, or 3D cultures,optionally where ambient conditions are controlled as in an incubator.

The term “substantially devoid of SANLCMs” as used herein means apopulation of cells comprising less than 30%, less than 25%, less than20%, or less than 15%, less than 10%, or less than 5% SANLCMs. In anembodiment, a population substantially devoid of SANLCMs is enriched forVLCMs.

The term “enriched for SANLCMs” as used herein means a population ofcells comprising at least 30%, at least 40%, at least 50%, at least 60%,or at least 70% up to 100% SANLCMs, for example in a day 20 cultureusing a method described in the Examples. The population “enriched forSANLCMs” can be further enriched or purified by isolating or purifyingcells using for example SIRPA^(pos) or SIRPA^(pos) NKX2-5^(neg) basedcell sorting. Isolated and/or purified SANLCMs based on SIRPA^(pos)CD90^(neg) can result in for example a population of cardiomyocyteswherein at least 60%, at least 70%, at least 80% or at least 90% up to100% are NKX2-5^(neg) SANLCM.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and suitably refers to humans.

The terms “treat”, “treating”, “treatment”, etc., as applied to a cell,include subjecting the cell to any kind of process or condition orperforming any kind of manipulation or procedure on the cell. As appliedto a subject, the terms refer to providing medical or surgicalattention, care, or management to an individual.

The term “treatment” as used herein as applied to a subject, refers toan approach aimed at obtaining beneficial or desired results, includingclinical results and includes medical procedures and applicationsincluding for example pharmaceutical interventions, surgery,radiotherapy and naturopathic interventions as well as test treatmentsfor treating cancer. Beneficial or desired clinical results can include,but are not limited to, alleviation or amelioration of one or moresymptoms or conditions, diminishment of extent of disease, stabilized(i.e. not worsening) state of disease, preventing spread of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

As used herein, the terms “administering”, “introducing” and“transplanting” are used interchangeably in the context of deliveringcells into a subject, by a method or route which results in at leastpartial localization of the introduced cells at a desired site.

The term “medium” as referred to herein is a culture medium forculturing cells containing nutrients that maintain cell viability andsupport proliferation and optionally differentiation. The medium maycontain any of the following in an appropriate combination: salt(s),buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum orserum replacement, and other components such as peptide growth factors,vitamins etc. For example, StemPro can be used as a medium.

The term “pluripotent stem cell” or “PSC” as used herein refers to acell with the capacity, under different conditions, to differentiate tomore than one differentiated cell type, and for example the capacity todifferentiate to cell types characteristic of the three germ celllayers, and includes embryonic stem cells and induced pluripotent stemcells. Pluripotent cells are characterized by their ability todifferentiate to more than one cell type using, for example, a nudemouse teratoma formation assay. Pluripotency is also evidenced by theexpression of embryonic stem (ES) cell markers. As used herein,pluripotent stems can include cell lines and induced pluripotent stemcells (iPSC) and embryonic stem cells (ESC).

In an embodiment, the term “embryonic stem cells” excludes stem cellsinvolving destruction of an embryo such as a human embryo.

As used herein, the terms “iPSC” and “induced pluripotent stem cell” areused interchangeably and refers to a pluripotent stem cell artificiallyderived (e.g., induced or by complete reversal) from a non-pluripotentcell, typically an adult somatic cell, for example, by inducingexpression of one or more genes (including POU4F1/OCT4 (Gene ID; 5460)in combination with, but not restricted to, SOX2 (Gene ID; 6657), KLF4(Gene ID; 9314), cMYC (Gene ID; 4609), NANOG (Gene ID; 79923),LIN28/LIN28A (Gene ID; 79727)).

The term “embryonic stem cell” is used to refer to the pluripotent stemcells of the inner cell mass of the embryonic blastocyst (see, forexample, U.S. Pat. Nos. 5,843,780, 6,200,806). Such cells can also beobtained from the inner cell mass of blastocysts derived from somaticcell nuclear transfer (see, for example, U.S. Pat. Nos. 5,945,577,5,994,619, 6,235,970). The distinguishing characteristics of anembryonic stem cell define an embryonic stem cell phenotype.Accordingly, a cell has the phenotype of an embryonic stem cell if itpossesses one or more of the unique characteristics of an embryonic stemcell such that that cell can be distinguished from other cells.Exemplary distinguishing embryonic stem cell characteristics include,without limitation, gene expression profile, proliferative capacity,differentiation capacity, karyotype, responsiveness to particularculture conditions, and the like.

The term “pharmaceutically acceptable carrier” as used herein includesessentially chemically inert and nontoxic compositions that do notinterfere with the effectiveness of the biological activity of thepharmaceutical composition. Examples of suitable pharmaceutical carriersinclude, but are not limited to, water, saline solutions, glycerolsolutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammoniumchloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), andliposomes. Such compositions should contain a therapeutically effectiveamount of the compound(s), together with a suitable amount of carrier soas to provide the form for direct administration to the subject.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins, andcell surface expression, including where applicable, but not limited to,for example, transcription, translation, folding, modification andprocessing. “Expression products” include RNA transcribed from a geneand polypeptides obtained by translation of mRNA transcribed from agene.

In understanding the scope of the present disclosure, the term“concentration” as used herein means a final concentration of asubstance such as for example BMP4, Activin A, retinoic acid in amedium. Unless indicated otherwise, the concentration is based on aweight/volume ratio.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. Finally, terms of degree such as “substantially”, “about”and “approximately” as used herein mean a reasonable amount of deviationof the modified term such that the end result is not significantlychanged. These terms of degree should be construed as including adeviation of at least ±5% of the modified term if this deviation wouldnot negate the meaning of the word it modifies.

In understanding the scope of the present disclosure, the term“consisting” and its derivatives, as used herein, are intended to beclose ended terms that specify the presence of stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.” Further, it is to be understood that “a,” “an,” and “the”include plural referents unless the content clearly dictates otherwise.The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

II. Methods and Products

Described herein are methods for producing and isolating a population ofcells enriched for- or substantially devoid-of SANLCMs. Components andconditions for specifying this cell type as well as markers formonitoring emergence of these cells are described.

An aspect includes a method of producing a population of cardiomyocytesenriched for sinoatrial node-like pacemaker cardiomyocytes (SANLCM) fromhuman pluripotent stem cells (hPSCs), the steps comprising:

-   -   a. incubating cardiovascular mesoderm cells in a cardiac        induction medium comprising a BMP component, optionally BMP4,        above a selected amount, and retinoic acid (RA), and optionally        one or more of a WNT inhibitor, optionally IWP2, VEGF; and an        activin/nodal inhibitor, optionally SB-431542; for a period of        time to generate cardiovascular progenitor cells that express        TBX18;    -   b. incubating the cardiovascular progenitor cells in a basic        medium comprising VEGF for a period of time to generate a        population of cardiomyocytes enriched for SANLCMs; and    -   c. optionally isolating the population of cardiomyocytes        enriched for SANLCMs using a cardiomyocyte-specific surface        marker, optionally wherein the marker is signal-regulatory        protein alpha (SIRPA), optionally wherein the isolated        population is SIRPA^(pos) CD90^(neg).

In some embodiments, the starting population is cardiovascular mesodermcells. Such cells, as shown in FIG. 5a express surface markersPDGRFalpha^((high)) and KDR^((low)). In addition, these cells expresssurface marker CD56. As shown in FIG. 5B, these cells express MESP1 andT(Brachyury) by Q-RT-PCR and can give rise to cTNT⁺ cardiomyocytes underconditions described herein and for example in Example 1.

As demonstrated in FIG. 12, addition of FGF inhibitor to cardiovascularmesoderm cells dramatically increases the proportion of SANLCMS whenassessed at day 20 of culture.

Accordingly in an embodiment, the cardiovascular mesoderm cells are alsotreated with FGF inhibitor for all or part of the cardiac inductionphase.

In embodiments for producing a population of cardiomyocytes enriched forSANLCM, the cardiovascular mesoderm cells are preferably incubated in acardiac induction medium comprising BMP4 above a selected amount, RA, aWNT inhibitor, VEGF, an activin/nodal inhibitor, and a FGF inhibitor.

In an embodiment, the FGF inhibitor is selected from PD 173074 (Torcis),SU 5402 (Torcis), and any other FGF receptor inhibitor or FGF signalinginhibitor.

Exemplary concentrations include 60 nM to 5 microMolar for PD173074, 5microMolar to 10 mM for SU 5402, or 1 microMolar to 1 mM for p38MAPKinhibitor SB203580. Any 1 nanoMolar increment between the stated rangesis also contemplated. For example the concentration of PD173074 can be61 nM, 62 nM, 63 nM etc

In an embodiment, the concentration of PD173074 includes or is selectedto be between 60 nM and 5 microMolar, for example, at least or about0.12 microMolar, at least or about 0.24 microMolar, at least or about0.5 microMolar, at least or about 0.75 microMoalar, at least or about 1microMolar, at least or about 2 microMolar, at least or about 3microMolar, at least or about 4 microMolar or about 5 microMolar. In anembodiment, the concentration of PD173074 is about 240 to about 960 nM.

In an embodiment, the cardiovascular mesoderm cells are incubated withthe FGF inhibitor for about 2 to about 7 days, optionally about 2 days,about 3 days, about 4 days or about 5 days.

The FGF inhibitor can be added between about 2.5 days and 5 days ofdifferentiation. The FGF inhibitor is added between about day 2.5 toabout day 5 which corresponds to the period when co-expression ofPDRGFalpha and CD56 is first detectable by flow cytometry and whenT(Brachyury) and MESP1 expression determined by Q-RT PCR reaches itsmaximum expression. As shown herein, both T(Brachyury) and MESP1 peakexpression around day 3 and then decrease—see FIG. 5B).

As demonstrated in FIG. 12, the method can be performed without a NKX2-5reporter when FGF inhibitor is added to the cardiac induction phase,permitting obtaining cultures that do not contain (or contain few)NKX2-5+ cells. These cultures contain predominantly SANLCM andnon-cardiomyocytes. Sorting for SIRPA+CD90− cells allows forpurification of SANLCM (as only few NKX2-5+ cells are present in theculture) from the non-cardiomyocytes.

Another aspect includes a method of producing a population ofcardiomyocytes substantially devoid of SANLCM from human pluripotentstem cells (hPSCs), the steps comprising:

-   -   a. incubating cardiovascular mesoderm cells in a cardiac        induction medium comprising one or more of a WNT inhibitor,        optionally IWP2, and VEGF; and optionally a FGF component and/or        an activin/nodal inhibitor, optionally SB-431542; for a period        of time to generate cardiovascular progenitor cells;    -   b. incubating the cardiovascular progenitor cells in a basic        medium comprising VEGF for a period of time to generate a        population of cardiomyocytes that are enriched for NKX2-5^(pos)        cTNT^(pos) and substantially devoid of SANLCMs; and    -   c. optionally isolating the population of cardiomyocytes that        are NKX2-5^(pos) cTNT^(pos) substantially devoid of SANLCMs        using a cardiomyocyte-specific surface marker optionally wherein        the marker is signal-regulatory protein alpha (SIRPA),        optionally wherein the isolated population is SIRPA^(pos)        CD90^(neg).

Addition of an activin/nodal inhibitor during the cardiovascularmesoderm cells for embodiments where a population substantially devoidof SANLCMs are desired, may be optionally used. For example any hESC orhIPSC cell line that has relatively high endogenous levels of activinsignaling is preferably treated with an activin/nodal inhibitor such asSB-431542.

In an embodiment, the cardiac induction medium comprises a WNT inhibitorand VEGF. In an embodiment, either a WNT inhibitor or VEGF can be used.As shown herein, inclusion of both components increases the efficiencyof generating NKX2-5pos cTNTpos cells. For example, the combination canresult in up to 80% of the culture comprising NKX2-5pos cTNTpos cells.

In an embodiment, the FGF component used to treat the cardiac mesodermcells is bFGF. Other FGF components as described herein can also beused.

In an embodiment, the concentration of bFGF in the cardiac inductionmedium is from about 0.25-100 ng/ml or any 0.1 ng/ml increment therebetween, for example about 1 ng/ml, about 10 ng/ml, about 20 ng/ml,about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70ng/ml, about 80 ng/ml, about 90 ng/ml, or about 100 ng/ml. In anembodiment, the FGF component is a FGF other than bFGF and is at aconcentration equivalent to from about 0.25-100 ng/ml or any 0.1 ng/mlincrement there between of bFGF.

In an embodiment, the cardiovascular mesoderm cells are incubated withthe FGF component for about 2 to about 7 days, optionally about 2 days,about 3 days, about 4 days or about 5 days.

As shown herein, cardiovascular progenitors can express TBX18 and cangive rise to SANLCM using methods described herein. Also cardiovascularprogenitors can express NKX2-5 and give rise to VLCM.

In an embodiment, the cardiovascular mesoderm cells are produced fromembryoid bodies, the method comprising:

incubating embryoid bodies in a mesoderm induction medium comprising aBMP component, optionally BMP4, and an activin component, optionallyActivin A, and optionally a FGF component, optionally bFGF, for a periodof time to generate cardiovascular mesoderm cells. Methods for producingcardiomyocyte populations including embryoid bodies and cardiovascularmesoderm cells are disclosed in PCT application no. PCT/CA2014/000687(entitled METHODS AND COMPOSITIONS FOR GENERATING EPICARDIUM CELLS)filed on Sep. 12, 2014 which is herein incorporated by reference in itsentirety.

Embryoid bodies can be obtained by incubating hPSCs in an embryoid bodymedium comprising a BMP component, optionally BMP4, optionally furthercomprising a Rho-associated protein kinase (ROCK) inhibitor, for aperiod of time to generate embryoid bodies.

In another embodiment, embryoid bodies used to obtain cardiovascularmesoderm cells can be obtained by incubating hPSCs in an embryoid bodymedium comprising a BMP component, optionally BMP4, optionally furthercomprising a Rho-associated protein kinase (ROCK) inhibitor, for aperiod of time to generate embryoid bodies.

In an embodiment, cardiovascular mesoderm cells can be obtained byincubating embryoid bodies in a mesoderm induction medium comprising aBMP component, optionally BMP4, and an activin component, optionallyActivin A and optionally a FGF component, optionally bFGF, for a periodof time to generate cardiovascular mesoderm cells.

Accordingly, another aspect includes a method of producing a populationof cardiomyocytes enriched for sinoatrial node-like pacemakercardiomyocytes (SANLCM) from human pluripotent stem cells (hPSCs), thesteps comprising:

-   -   a. incubating the hPSCs in an embryoid body medium comprising a        BMP component, optionally BMP4, optionally further comprising a        Rho-associated protein kinase (ROCK) inhibitor, for a period of        time to generate embryoid bodies;    -   b. incubating the embryoid bodies in a mesoderm induction medium        comprising a BMP component, optionally BMP4, and an activin        component, optionally Activin A and optionally a FGF component,        optionally bFGF, for a period of time to generate cardiovascular        mesoderm cells;    -   c. incubating the cardiovascular mesoderm cells in a cardiac        induction medium comprising a BMP component, optionally BMP4,        above a selected amount, and retinoic acid (RA), and optionally        one or more of a FGF inhibitor, a WNT inhibitor, optionally        IWP2, VEGF and an activin/nodal inhibitor, optionally SB-431542;        for a period of time to generate cardiovascular progenitor cells        that express TBX18, wherein the cardiovascular mesoderm cells        are preferably incubated with the FGF inhibitor and which FGF        inhibitor is provided for all or part of the cardiac induction        phase;    -   d. incubating the cardiovascular progenitor cells in a basic        medium comprising VEGF for a period of time to generate a        population of cardiomyocytes enriched for SANLCMs; and    -   e. optionally isolating the population of cardiomyocytes        enriched for SANLCMs using a cardiomyocyte-specific surface        marker, optionally wherein the marker is signal-regulatory        protein alpha (SIRPA), optionally wherein the isolated        population is SIRPA^(pos) CD90^(neg).

As demonstrated herein, any human pluripotent stem cell line can beused, including for example embryonic stem cell lines and inducedpluripotent stem cells (iPSCs) derived for example from patient blood orskin samples. Methods for producing iPSCs are known in the art iPSCs canbe derived from multiple different cell types, including terminallydifferentiated cells. iPSCs have an ES cell-like morphology, and expressone or more key pluripotency markers known by one of ordinary skill inthe art, including but not limited to Alkaline Phosphatase, SSEA3,SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3,Cyp26a1, TERT, and zfp42. Examples of methods of generating andcharacterizing iPSCs may be found in, for example, U.S. PatentPublication Nos. US20090047263, US20090068742, US20090191159,US20090227032, US20090246875, and US20090304646, the disclosures ofwhich are incorporated herein by reference. Generally, to generateiPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4,SOX2. KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram thesomatic cells to become pluripotent stem cells.

As shown herein, hPSC lines can be used to produce cultures enriched forSANLCMs which can be further enriched/purified using cell sorting. Forexample, in embodiments where FGF inhibitors are used to increase theproportion of SANLCMs (which reduces the NKX2-5+ cTNT+ cardiomyocytepopulation) selection of cardiomyocytes from these cultures by Flowassisted cells sorting (FACS) for SIRPA⁺ and CD90⁻ cells achievescultures highly enriched in SANLCMs (e.g. 70-90%).

In an embodiment, the hPSCs are induced pluripotent stem cells (iPSCs).In an embodiment, the hPSCs are human embryonic stem cells (hESCs).

In other embodiments, mammalian PSCs are used in the methods describedherein, including for example but not limited to rodent such as mouseand rat PSCs, non-human primate PSCs and pig PSC.

In an embodiment, the hESCs are H7 cells, HES2 cells or HES3 cells. Inan embodiment, the iPSCs are MSC-iPS1 cells. In a further embodiment,the iPSCs are prepared from cells obtained from a subject.

iPSCs can be produced according to known methods such as the methodsdisclosed in U.S. Pat. Nos. 8,278,104 and 8,058,065 which are hereinincorporated by reference

In some embodiments, hPSCs (or mammalian PSCs) comprising a reporterconstruct are used to monitor development of cardiac populations. In anembodiment, the hPSCs are hPSCs comprising a NKX2-5 reporter construct.

In an embodiment, the BMP component is BMP4. In another embodiment, theBMP component is BMP2.

In another embodiment, isolating or purifying the population ofcardiomyocytes enriched for SANLCMs comprises:

-   -   a. isolating SIRPA marker positive cardiomyocytes; and/or    -   b. selecting cardiomyocytes negative for NKX2-5 expression        and/or negative for CD90 expression.

In another embodiment, isolating the population of cardiomyocytessubstantially devoid of SANLCMs comprises:

-   -   a. isolating SIRPA marker positive cardiomyocytes; and/or    -   b. selecting cardiomyocytes positive for NKX2-5 expression        and/or negative for CD90 expression.

As demonstrated herein, to isolate a population enriched forcardiomyocytes, a cardiomyocyte cell-surface marker can be used. Forexample, SIRPA is a cardiomyocyte specific cell-surface marker that canbe used for isolating cardiomyocytes derived from human pluripotent stemcells.¹⁰ For instance, cells such as stem cell differentiation culturescan be sorted with an antibody against SIRPA. In Dubois et al.¹⁰, it wasshown that cell sorting with an antibody against SIRPA yieldedpopulation of up to 98% cardiac troponin T (cTnT)-positive cells.

NKX2-5 or homeobox protein NKX2-5 is a transcription factor involved inthe regulation of cardiomyocyte formation. It is expressed in somecardiomyocyte subtypes such as ventricular cardiomyocytes but not inothers, such as SANLCMs.

It is demonstrated herein that selecting NKX2-5 negative cells enrichesfor SANLCMs and selecting NKX2-5 positive cells from cultures treatedwith cardiac induction medium comprising FGF as described hereinenriches for ventricular-like cardiomyocytes. Selecting for NKX2-5 cellscan be used for example in methods using a NKX2-5 reporter assay.Measuring NKX2-5 expression levels can also be used to confirm that apopulation obtained is the desired population and/or to confirm theamount of undesired cells. For example, an aliquot of a cardiomyoctyepopulation treated according to methods described herein for enrichingfor SANLCMs can tested to confirm that level of NKX2-5 expression is low(e.g. as in an unpurified population) or virtually absent (e.g. as in apopulation that is purified for SIRPApos CD90neg cells). Similarly, analiquot of a cardiomyoctye population treated according to methodsdescribed herein for producing a population substantially devoid ofSANLCMs (e.g. using VEGF and IWP2 in the cardiac induction phase) can betested to confirm that the level of NKX2-5 expression is high.

As demonstrated NKX2-5 negative selected cardiomyocytes arecardiomyocytes enriched for SANLCMs.

In another embodiment, NKX2-5 positive selected cardiomyocytes arecardiomyocytes enriched for ventricular-like cardiomyocytes (VLCMs).

For example, a NKX2-5 reporter construct can be used to identifycardiomyocytes such as stem cell derived cardiomyocytes. As shown inElliott et al.¹⁶, sequences encoding enhanced GFP were introduced intothe NKX2-5 locus by homologous recombination and NKX2-5:GFP positivehESCs differentiated into cardiac progenitor cells.

Fluorescence detection techniques such as immunofluorescence analysiscan be used to select or determine NKX2-5 eGFP expressing cells. Forexample, the presence of fluorescence in reporter cells is indicativethat the cell expresses NKX2-5. For example, the absence of fluorescenceis indicative that the cell does not express NKX2-5 or does not expresshigh levels of NKX2-5. Other NKX2-5 reporter constructs can also beused.

Cells can be isolated for example using flow cytometry (e.g. FACS) basedon marker expression (e.g. cell surface markers) and/or when usingfluorescent based reporters.

Other methods can be used with other reporters, for example cellscomprising an antibiotic resistant reporter gene can be isolated basedon their antibiotic resistance to for example Geneticin®, Puromycin orHygromycin B.

Other reporter constructs can be used to produce a population ofcardiomyocytes enriched for SANLCMs. For example, SAN specific markerscan be used. In an embodiment, the reporter construct is a SHOX-2reporter construct. In another embodiment, the reporter construct is aTBX18 reporter construct.

While it is not necessary to use a NKX2-5 reporter construct to producea population of cardiomyocytes enriched for SANLCMs, using an hPSC cellline comprising a NKX2-5 reporter or introducing a NKX2-5 reporterconstruct in a stem cell such as a hPSC line can be used to monitorand/or optimize the SANLCM differentiation protocol, e.g. the presenceof NKX2-5 expression can be measured and the protocol can be adjustedaccordingly until a absence and/or minimum desirable presence of NKX2-5expression is detected by for example immunofluorescence analysis.

Alternatively, the method of producing a population of cardiomyocytesenriched for SANLCMs described above can comprise the use of an antibodyspecific for NKX2-5. For example, an antibody to NKX2-5 such as rabbitanti-human NKX2-5 (1:800, Cell Signaling Technology) can be used.

For example, an aliquot of a cell culture can be assessed to measure thelevel of NKX2-5 and/or one or more markers described herein to assessfor example that a desired level or number of cells are expressing amarker associated with a stage described herein. The cell culture can betested at one or more stages and/or upon completion to measure forexample the number of SANLCMs produced. The level of one or more markers(cell surface or intracellular) can be measured using immuno-basedmethods for example, flow cytometry, including FACS, or mRNA expressionbased methods such as quantitative RT-PCR,

In an embodiment, the population of cardiomyocytes enriched for SANLCMscomprises at least or about 30% of SANLCMs, at least or about 50% ofSANLCMs, at least or about 70% of SANLCMs, or at least or about 90% ofSANLCMs.

In an embodiment, the population of cardiomyocytes enriched for VLCMscomprises at least or about 15% of VLCMs, at least or about 20% ofVLCMs, at least or about 30% of VLCMs, at least or about 50% of VLCMs,at least or about 70% of VLCMs, or at least or about 90% of VLCMs.

FIG. 1A illustrates a scheme of the developmentally staged protocol forthe hESC differentiation into cardiomyocytes. In an embodiment, thehPSCs are incubated in the embryoid body medium at day 0 of thedifferentiation process. In an embodiment, the embryoid bodies areincubated in the mesoderm induction medium from about day 1 to about day3 of the differentiation process. In an embodiment, the cardiovascularmesoderm cells are incubated in the cardiac induction medium from aboutday 3 to about day 5 of the differentiation process. In an embodiment,the cardiovascular progenitor cells are incubated in a basic medium fromabout day 5 to about day 20 of the differentiation process.

In an embodiment, the hPSCs are incubated in the embryoid body medium togenerate embryoid bodies from about 6 hours to about 2 days, optionally18 hours.

In an embodiment, the embryoid bodies are incubated in the mesoderminduction medium to generate cardiovascular mesoderm cells for about 1to about 4 days, optionally 2 days.

In an embodiment, the cardiovascular mesoderm cells are incubated in thecardiac induction medium to generate cardiovascular progenitor cells forabout 1 to about 4 days, optionally 2 days.

In an embodiment, the cardiovascular progenitor cells are incubated inthe basic medium to generate cardiomyocytes for about 4 or more days,optionally about 4, about 5, about 9, about 15 or about 20 days. In anembodiment, the cardiovascular progenitor cells are incubated in thebasic medium to generate cardiomyocytes from about 4 days to about 20days or any number of days between 4 days and 20 days. In an embodiment,the cardiovascular progenitor cells are incubated in the basic medium togenerate cardiomyocytes for over 20 days.

In an embodiment, the mesoderm induction medium comprises BMP4 at aconcentration ranging between about 0.5 ng/mL to about 5 ng/mL, betweenabout 0.5 ng/mL to about 3 ng/mL, between about 0.5 ng/mL to about 8ng/mL, between about 2 ng/mL to about 10 ng/mL, between 0.5 ng/mL toabout 10 ng/mL, or between 0.5 ng/mL to about 20 ng/mL. Theconcentration can be or range from any 0.1 ng/mL increment between about0.5 ng/mL up to about 20 ng/mL.

In an embodiment, the mesoderm induction medium comprises BMP4 at aconcentration of about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4ng/mL, about 5 ng/mL, about 8 ng/mL, or about 10 ng/mL, or up to about20 ng/mL, optionally about 3 ng/mL.

In an embodiment, the mesoderm induction medium comprises Activin A at aconcentration ranging between about 0.5 ng/mL to about 3 ng/mL, betweenabout 0.5 ng/mL to about 4 ng/mL, between about 0.5 ng/mL to about 5ng/mL, between about 1 ng/mL to about 10 ng/mL, between about 1 ng/mL toabout 20 ng/mL, between 0.1 ng/mL to about 10 ng/mL, or between 0.1ng/mL to about 20 ng/mL. The concentration can be or range from any 0.1ng/mL increment between about 0.1 ng/mL up to about 20 ng/mL.

In another embodiment, the mesoderm induction medium comprises Activin Aat a concentration of about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about5 ng/mL, about 8 ng/mL, about 10 ng/mL, or about 20 ng/mL, optionallyabout 2 ng/mL.

In an embodiment, the mesoderm induction medium comprises BMP4 at aconcentration of about 3 ng/mL and Activin A at a concentration of about2 ng/mL.

BMP4 signaling plays an important role in the specification of thepacemaker population at the mesoderm stage (around day 3) and as such anadequate dosing and time of treatment with BMP4 is important in thegeneration of cardiomyocytes enriched for SANLCMs. It was previouslyshown that blocking BMP signaling and exposure to high levels of BMP4resulted in a significant reduction of cardiomyocytes. As shown in FIG.2B-D and FIG. 7B, lower levels of BMP4 lead to an increase in theproportion of SANLCMs.

In an embodiment, the cardiovascular mesoderm cells are treated withcardiac induction medium comprising BMP4 for about 1 day to about 4days, optionally 2 days, at concentrations of about 0.5 ng/mL, about 1.0ng/mL, about 1.5 ng/mL, about 2.0 ng/mL, about 2.5 ng/mL, about 3.0ng/mL, about 5.0 ng/mL, about 10.0 ng/mL, about 20.0 ng/mL, about 30.0ng/mL, about 40.0 ng/mL, about 50.0 ng/mL, about 60.0 ng/mL, about 70.0ng/mL, or about 80.0 ng/mL, optionally 2.5 ng/mL.

As shown in FIG. 3, RA signaling also impacts the development ofSANLCMs. While adding RA at day 2 of the differentiation process blockedthe generation of cardiomyocytes, adding RA at later times had no effecton total cardiomyocytes or SANLCM frequency. It is shown herein thataddition of RA between days 3 and 5 significantly upregulates theposterior cardiomyocyte marker TBX5 and the SAN marker SHOX2. It isdemonstrated that RA signaling enhances the pacemaker phenotype in SANprogenitors (FIG. 3H).

In an embodiment, the cardiovascular mesoderm cells are treated withcardiac induction medium comprising RA for about 1 day to about 4 days,optionally 1 day, at concentrations of about 50 ng/mL, about 100 ng/mL,about 150 ng/mL, about 200 ng/mL, about 300 ng/mL, about 400 ng/mL,about 500 ng/mL, or about 1000 ng/mL, optionally 150 ng/mL or anyconcentration or range from any 0.1 ng/mL increment between about 50ng/mL up to about 1000 ng/mL (alternatively about 100 nM to about 3 mMor any 1 nM increment between 100 nM and 3 mM.

In an embodiment, the RA is all-trans RA (Sigma R2625) or 9-cis RA(Sigma R4643) at a concentration ranging between about 20 ng/ml to about1000 ng/ml, optionally at a concentration of about 150 ng/mL.

In another embodiment, the RA is a RA analog. In an embodiment, the RAanalog is AM580, a selective RARα agonist (Tocris 0760) at aconcentration ranging from about 1 ng/ml to about 500 ng/ml, optionallyat a concentration of about 18 ng/mL. In an embodiment, the RA analog isAC55649, a selective RARβ agonist (Tocris 2436) at a concentrationranging from about 10 ng/ml to about 1000 ng/ml, optionally at aconcentration of about 80 ng/mL. In an embodiment, the RA analog isCD437, a selective RARγ agonist (Tocris 1549) at a concentration rangingfrom about 50 ng/ml to about 5000 ng/ml, optionally at a concentrationof about 600 ng/mL.

It is shown herein that combined treatment of cardiovascular mesodermcells with BMP4 and RA between days 3 and 5 increases the SANLCMdifferentiation process. As shown in FIG. 3, combined RA/BMP4 signalingsignificantly increased the expression of the pacemaker ion channelgenes HCN4 and HCN1 (FIG. 3F) but not the AVN specific genes TBX2 andMSX2. Also, combined RA/BMP4 signaling significantly increased thebeating rate of the cells (FIG. 2G). Further, treatment of RA alone andin combination of BMP4 increased the expression of TBX5, SHOX2 and ISL1(FIG. 3E) but decreased the expression of MYL2 (a ventricular marker).

In another embodiment, the cardiovascular mesoderm cells are treatedwith BMP4 and RA. In yet another embodiment, the cardiovascular mesodermcells are treated with BMP4 at a concentration of about 2.5 ng/mL and RAat a concentration of about 150 ng/mL.

In another embodiment, the cardiovascular mesoderm cells are treatedwith BMP2 and RA. In some embodiments, the cardiovascular mesoderm cellsare treated with RA (e.g. in the absence of BMP component).

As demonstrated in FIG. 12, treating cardiovascular mesoderm cells withFGF inhibitor increases the proportion of SANLCMs. Without wishing to bebound by theory, inhibition FGF signaling removes the NKX2-5^(pos)cTNT^(pos) cardiomyocyte population.

Further, as shown in FIG. 12A treating cardiovascular mesoderm cellswith FGF decreases the proportion of SANLCMs and generates NKX2-5poscardiomyocyte cultures that are substantially devoid of SANLCMs. Withoutwishing to be bound by theory, FGF signaling removes theNIKX2-5^(neg)cTNT^(pos) cardiomyocyte population and any pacemakercells.

Accordingly in another embodiment, the cardiovascular mesoderm cells aretreated with BMP2, RA and FGF inhibitor, optionally PD173074. In otherembodiments the cardiovascular mesoderm cells are treated with BMP2, RAand FGF, optionally bFGF.

In an embodiment, the cardiovascular mesoderm cells are incubated atleast until they express a desired level of MESP1, PDGFRalpha and/or KDRand/or a desired proportion of cells expressing MESP1, PDGFRalpha and/orKDR.

MESP1, KDR and/or PDGFRalpha can be used to monitor the development ofcardiovascular mesoderm cells. For example, using immune-based methods,the expression of KDR can be monitored for example by FACs using anantibody specific for KDR. The expression of PDGFRalpha can be monitoredby FACS using an antibody specific for PDGFRalpha. These markers can bymonitored using flow cytometry. For example, the expression of MESP1,KDR and PDGFRalpha can be monitored and/or cells expressing thesemarkers can be isolated using cell sorting, for example FACS or otherimmunostaining methods.

In another embodiment, the cardiovascular progenitor cells and/orcardiomyocytes are incubated until they express a desired level ofcardiac troponin T (cTnT).

cTNT can be used to monitor the development of cardiomyocytes. Theexpression of cTNT can be used to monitor using an antibody specific forcTNT, for example anti-cardiac isoform of cTNT (clone 13-11; 1:2000,Thermo Fischer). The expression of cTNT can be monitored using flowcytometry, for example FACS. The expression of cTNT can also bemonitored by immunostaining.

CD90 is a cell surface mesenchymal marker that can be used to isolatenon-myocyte populations in differentiation cultures. In an embodiment,CD90 can be used to deplete non-myocytes and/or to isolatecardiomyocytes.

In another embodiment, cardiomyocyte subtypes SANLCMs and VLCMs can besorted, for example by FACS, according to NKX2-5, SIRPA and CD90expression. For example, as shown in FIGS. 1D and 5C, cardiomyocytepopulations are sorted for NKX2-5⁺SIRPA⁺CD90⁻ and NKX2-5⁻SIRPA⁺CD90⁻populations at day 20 of differentiation.

TBX18, SHOX2 and TBX3 are SAN specific markers. In an embodiment,cardiomyocytes enriched for SANLCMs express an increased level of TBX18,SHOX2 and/or TBX3 compared to cardiovascular mesoderm cells not treatedwith the BMP component and RA.

MYL2 and IRX4 are ventricular cardiomyocyte specific markers. In anembodiment, cardiomyocytes enriched for SANLCMs express lower levels ofMYL2 and IRX4 compared to cardiovascular mesoderm cells not treated withthe BMP component and RA.

It is demonstrated herein that SANLCMs have significantly fasterspontaneous action potential rates compared to VLCMs (FIG. 4A-B).

In an embodiment, the SANLCMs have a minimum or average spontaneousbeating rate of at least 50 beats per minute (BPM), at least 60 BPM, atleast 80 BPM, at least 90 BPM, at least 100 BPM, at least 110 BPM, atleast 120 BPM, at least 140 BPM at least 160 BPM at least 180 BPM, up toabout 200 BPM.

Beating rates can be measured to assess the functionality of SANLCMs aspacemakers. As described in Example 1, the functionality of SANLCMs canbe determined by isolating ventricular-like cardiomyocytes (VLCMs),optionally by FACS, and forming electrically integrated monolayers onfor example a multi-electrode array. Aggregates of SANLCMs are placed onthe VLCM monolayers and cultured to allow electrical integration. It isshown that the SANLCM aggregate can stably initiate and propagateelectric activity through the adjacent monolayer and as a resultincrease the beating frequency from 63.1±2.5 to 112.5±18.5 bpm (Table2).

Another aspect includes a method of isolating SANLCMs or VLCMs from apopulation of cardiomyocytes comprising a) producing a population ofcardiomyocytes from hPSCs comprising a NKX2-5 reporter construct,optionally according to a method described herein and b) selectingNKX2-5 negative or positive cardiomyocytes.

In an embodiment, the NKX2-5 reporter construct is a fluorescent NKX2-5reporter construct.

In an embodiment, the fluorescent reporter comprises a GFP (optionallyenhanced GFP) reporter gene. Other fluorescent proteins as well asnon-fluorescent markers can be used.

In an embodiment, one or more or all of the steps of a method describedherein are performed in vitro.

A further aspect includes an isolated population of cardiomyocytesenriched for SANLCMs comprising at least or about 30% of SANLCMs, atleast or about 50% of SANLCMs, at least or about 60% of SANLCMs, atleast or about 70% of SANLCMs, at least or about 80% of SANLCMs or atleast or about 90% of SANLCMs, obtained according to a method describedherein.

A further aspect includes an isolated population of cardiomyocytesenriched for VLCMs comprising at least or about 15% of VLCMs, at leastor about 20% of VLCMs, at least or about 30% of VLCMs, at least or about50% of VLCMs, at least or about 60% of VLCMs, at least or about 70% ofVLCMs, at least or about 80% of VLCMs or at least or about 90% of VLCMs,obtained according to a method described herein.

Another aspect includes a SANLCM or VLCM comprising a NKX2-5 reporterconstruct or a population of SANLCMs comprising a NKX2-5 reporterconstruct obtained according to the method herein described.

A further aspect includes various uses of the isolated population ofcardiomyocytes, for example cardiomyocytes substantially devoid (VLCM)or enriched for SANLCMs. Uses include transplant, for example for invivo pacemaking (SANLCM) or for remuscularization after myocardialinfarction (VLCM) in a subject, disease modelling and testing candidatedrugs. Other uses include studying the safety pharmacology testing ofdrugs (not developed to treat heart conditions) for potential sideeffects on SANLCM and VLCM, the development of SAN pacemaker or VLCMcells using the human stem cell system, and studying the physiology ofhuman SAN pacemaker and VLCM as healthy human samples are not readilyavailable.

Accordingly a further aspect is a method of identifying a candidate drugcomprising:

-   -   a. generating a SANLCM according to a method described herein;    -   b. contacting the SANLCM with a candidate test drug;    -   c. measuring the beat rate, action potential characteristics        and/or ion currents of the SANLCM;    -   d. comparing the beat rate, the action potential characteristics        and/or ion currents of the SANLCM to a control SANLCM not        treated with the candidate test drug; and    -   e. selecting the candidate test drug which modulates the beat        rate and/or action potential compared to the control cell as the        candidate drug.

In an embodiment, the SANLCM is comprised in a population ofcardiomyocytes enriched for SANLCMs.

Various methods known in the art can be used to measure the actionpotential in a cell. In an embodiment, the action potential in a cell ismeasured using low throughput patch clamp (as shown in FIG. 4 A-E).

In an embodiment the electric signals in a cell are measured usingmulti-electrode arrays (MEA) (see FIG. 4F and FIG. 10). The MEA systemis a medium throughput system which records field potentials that can beindicative of drug effects on ion-channels/currents.

An in-vitro pacemaking assay comprising SANLCMs aggregates and a VLCMmonolayer can be used to test whether a candidate drug can disrupt theability of SANLCMs to pace a VLCM monolayer. For example, VLCM cells areplaced on MEAs and cultured until formation of an electrical integratedmonolayer. Aggregates of SANLCMs are then placed on the VLCM monolayerand following electrical integration of SANLCM aggregates and VLCMmonolayer, field potentials are recorded.

In an embodiment, the in-vitro pacemaking assay is used as a safetypharmacology screen.

In an embodiment, the candidate drug is for treating diseases affectingthe SAN, including Sick Sinus Syndrome (SSS), bradycardia, tachycardia,sinus node arrest/block caused by aging (mostly fibrosis of the sinusnode) or genetic disorder.

Genetic disorders causing SSS described include for example SCN5a (GeneID: 6331) sodium channel mutations, HCN4 (Gene ID:10021) mutation, Cx40(Gene ID: 2702) mutation, Myh6 (Gene ID:4624) mutations and Ankyrin-B(Gene ID: 287) mutations.

Disease modeling can be accomplished by preparing SANLCM from a subjectwith a disease condition or disorder affecting pacemaker cells.

Accordingly in an embodiment, the hPSCs are induced hPSCs prepared froma subject with a suspected disease, condition or disorder affectingpacemaker cells or VLCMs cells. The cells can be used to test drugcandidates and/or for molecular, genomic, proteomic, physiological(including action potentials, ion currents) or other analyses.

In an embodiment, the use or method is for in vivo pacemaking. As shownin Example 2, the isolated population of cardiomyocytes enriched forSANLCMs and/or the substantially pure population of SANLCMs can be usedfor in vivo transplantation to confer pacemaker capacity.

In an embodiment, the use or method is for treatments requiringpopulations of cells free of pacemaker cells including for exampleventricular cell transplantation for remuscularization after myocardialinfarction.

The ability of the cells to function as an in vivo pacemaker was testedby transplanting cell aggregates into the apex of a rat heart. The heartbeat was first decreased pharmacologically to resemble that of a humanheart beat. It was shown ex-vivo, that rat hearts receiving the SANLCMtransplant displayed a significantly faster ventricular ectopic rhythmafter induction of atrioventricular (AV) block.

Cardiomyocytes for example enriched for SANLCMs or SANLCMs can beintroduced to the heart by a minimal invasive method using acatheter-based approach. The catheter can be inserted via the femoral,subclavian, jugular or axillary vein by endocardial transplantationapproach into the ventricle, atria or SAN region. Alternatively, thecells can be transplanted into the ventricle, atria or SAN region byepicardial approach using a needle inserted through the chest. In bothapproaches, Fluoroscopy (X-Ray based method) or 3D Mapping can be usedto guide the catheter/needle to the intended injection site.

The ability of the cells to function as a pacemaker was also assessed exvivo. Ten days post-transplantation, hearts were harvested andelectrocardiographic recordings and fluorescent voltage imaging wereperformed, as shown in Example 2. Optical mapping confirmed that the newectopic rhythm was initiated from the SANLCM transplantation site.

Accordingly a further aspect includes a method of treating a subject inneed thereof comprising administering to the subject a population ofcardiomyocytes enriched for SANLCM or SANLCM to the subject, a cell orcomposition described herein, or a biological pacemaker (e.g. 3D and/orcomprising endothelial cells, mesenchymal stem cells and/or smoothmuscle cells and optionally extracellular matrices that are used to form3D tissues).

Similarly, NKX2-5⁺ cardiomyocyte cultures that are free of SANLCMs canbe used in cell therapy approaches that require populations free ofpacemaker cells, for example for ventricular cell transplantation forremuscularization after myocardial infarction.

Accordingly a further aspect includes a method of treating a subject inneed thereof comprising administering to the subject a population ofcardiomyocytes substantially devoid of SANLCM to the subject, a cell orcomposition described herein.

Uses of the cell populations for treating a subject in need thereof asdescribed herein are also envisioned.

Administration to a subject of a population of cardiomyocytes, includingfor example cardiomyocytes enriched for SANLCMs or consisting of SANLCMsor substantially devoid of SANLCMs can be done for example by the use ofESC-derived (HLA class matched, not necessarily patient specific) cellsor for example by the use of iPSC-derived (patients' own cells) SANLCMsor VLCMs. The cells can be injected via a catheter (see above) as singlecells or small aggregates into the left or right ventricular wall orapex of the heart.

In addition, prior to in vivo use, it is possible to confirm thecardiomyocyte subtype and/or to ensure the desirable amount of SANLCMsor VLCMs have been obtained.

As described above, this can be accomplished by taking an aliquot of thecell sample and testing the sample for SAN markers (e.g. Shox2, Tbx18,Tbx3, ISL1) or other markers or their absence including NKX2-5expression using for example an antibody specific to NKX2-5 such asrabbit anti-human NKX2-5. This protocol can be applied as a qualitycontrol for the SANLCM differentiation prior to in vivo use of thecells.

For example, a small aliquot of the cells can be taken to test for anacceptable number or density of SANLCMs.

A further aspect is a composition comprising an isolated population ofcardiomyocytes, for example enriched for or substantially devoid ofSANLCMs and/or isolated SANLCMs or VLCMs and a pharmaceuticallyacceptable carrier. In an embodiment, the isolated population comprisescells comprising a NKX2-5 reporter such as NKX2-5 GFP reporter. In anembodiment, the isolated population is a clonal population derived froman ESC cell line or an iPSC.

As indicated by their names the SANLCM are SAN like CM and the VLCM areV like CM. They may represent a fetal development stage and thereforemay have one or more differences than adult SANCM and VCM. SANLCM forexample may beat faster—for example around 120 bpm, or around 150bpm—than an adult SAN pacemaker cell which beats around 60-100 bpm. Inan embodiment, the VLCM described herein may express a lower level ofKir2.1 ion-channel, higher level of HCN4, HCN2, and/or may have immaturecalcium handling properties compared to adult ventricularcardiomyocytes.

Yet a further aspect is a biological pacemaker comprising an isolatedpopulation of cardiomyocytes enriched for SANLCMs and/or isolatedSANLCMs and a pharmaceutically acceptable carrier.

In the engineering of a biological pacemaker, three dimensional (3D)tissues can be used to ensure vascularization and potential innervationof the grafted cells.

In an embodiment, the biological pacemaker further comprises endothelialcells, mesenchymal stem cells and/or smooth muscle cells and optionallyextracellular matrices that are used to form 3D tissues.

For example, Tolloch et al.³³ used human embryonic stem cells and humaninduced pluripotent stem cell-derived cardiomyocytes and developed acollagen-based, bio-engineered human 3D cardiac tissue construct in aself-organizing co-culture with endothelial and stromal cells.

In an embodiment, markers herein disclosed such as ventricular specificmarkers (e.g. MYL2 and IRX4) and SAN specific markers (e.g. TBX18, TBX3,SHOX2 and ISL1) can be detected using any one of the primer sequences inTable 3.

As mentioned herein VLCM populations can be produced by activating FGFsignaling to generate cultures of NKX2-5^(pos) cardiomyocytes. In somemethods, the methods for producing and/or isolating VLCM comprise usinga reporter construct. For example, an embodiment provides a method ofisolating ventricular-like cardiomyocytes from a population ofcardiomyocytes comprising a) producing a population of cardiomyocytesfrom PSCs comprising a NKX2-5 reporter construct as described herein andb) selecting NKX2-5 positive cardiomyocytes and/or removing NKX2-5negative cardiomyocytes.

Methods of producing VLCM cells are known and described for example inthe Witty A. D. et al. Generation of the epicardial lineage from humanpluripotent stem cells. Nature Biotechnology, doi:10.1038/nbt.3002(2014) which is incorporated herein by reference. The method describedin Witty et al can for example be enhanced by addition of a FGFcomponent during the cardiac induction phase

It is demonstrated herein that VLCM cells can be isolated and/or removedfrom a population of cardiomyocytes according to NXK2-5 reporterexpression.

For example, cells comprising a NKX2-5 reporter construct that arepositive for reporter protein expression can be isolated or depletedfrom a population of cardiomyocytes generated according to a methoddescribed herein.

A further aspect includes the use of an isolated population ofcardiomyocytes enriched for VLCMs and/or a population of VLCMs depletedof NKX2-5 expressing cells for in vivo transplanting in a subject.

It is demonstrated herein that VLCMs have reduced pacemaker capacity(FIG. 11A). VLCMs prepared from hPSCs depleted of SANLCMs may have somebenefits. For example, depleting SANLCMs could reduce arrhythmias upontransplantation of VLMCs, for example for ventricular celltransplantation for remuscularization after myocardial infarction.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

Methods

hPSC Maintenance and Differentiation

Human pluripotent stem cell lines (Hes3 NKX2-5:GFP)¹⁰ H7 (HESC lineapproved by NIH registration number 0061³² and MSC-iPS1¹¹ were culturedas described¹². For differentiation into the cardiac lineage theestablished protocol⁶ was used with the following modifications (FIG.1A). 80% confluent hPSCs cultures were dissociated into single cells,suspended in StemPro-34 Media containing 1 ng/ml BMP4 and 10 μM ROCKinhibitor and incubated for 18 h on an orbital shaker to generateembryoid bodies (EBs). The next day (day 1 of differentiation) the EBswere transferred to mesoderm induction media: StemPro-34 containing 10ng/ml BMP4, 6 ng/ml ActivinA, 5 ng/ml bFGF. At day 3 of differentiationthe EBs were washed once using IMDM and suspended in cardiac inductionmedia: StemPro-34 containing 0.5 μM IWP2, 10 ng/ml VEGF, and optionally5.4 μM SB-431542 (SB, Activin/Nodal/TGFβ inhibitor). At day 5 ofdifferentiation EBs were switched to basic media: Stempro containing 5ng/ml VEGF. Media was supplemented with VEGF until day 12 ofdifferentiation and changed every 4 days. All cytokines were purchasedfrom R&D systems if not indicated otherwise. EBs were cultured under lowoxygen conditions 5% CO₂, 5% O₂, 90% N₂ until day 12 and at 5% CO₂,normal air conditions for the rest of the culture period.

Pacemaker (SANLCM) Optimized Protocol

EBs were dissociated into single cells at the mesoderm stage (day 3 ofdifferentiation) using TrypLE (Life Technologies) with the rational toensure efficient cytokine supply to each cell. Cells were placed into 96well low cluster plates at 80,000 cells/well with the indicatedcytokines and 5 μM ROCK inhibitor for re-aggregation into EBs (FIG. 2A).At day 5 of differentiation EBs were transferred to 24 well low clusterplates and continued to be cultured in basic media as described above.

For efficient specification of SANLCM pacemaker, mesoderm was inducedwith the 3B/2A condition (3 ng/ml BMP4, 2 ng/ml ACTA) at day 1 ofdifferentiation. At day 3 EBs were dissociated as described above andtreated with 2.5 ng/ml BMP4 and 0.5 μM RA in addition to standardcardiac induction media.

For the generation of SANLCM cultures free of NKX2-5⁺ cardiomyocytesmesoderm was induced with the 3B/2A condition (3 ng/ml BMP4, 2 ng/mlACTA) at day 1 of differentiation. For this protocol EBs were keptintact and treated at day 3 with 2.5 ng/ml BMP4, 0.5 μM RA and 960 nMFGF inhibitor (PD 173074, Tocris) at day 3 or day 4 in addition tostandard cardiac induction media.

Flow Cytometry and Cell Sorting

For the dissociation of day 3-day 12 EBs TrypLE was used. Day 12-30 EBswere incubated in HANKs buffer containing 1 mg/ml Collagenes type 2(Worthington) with gentle shaking over night at room temperature, priorto TypLE dissociation. Cells were stained at a concentration of 5×10⁶cells/ml with anti-PDGFRα-PE (1:20) anti-KDR-APC (1:10), (R&D Systems),anti-SIRPA-PeCy (clone SE5A5, 1:1000, Biolegend), anti-CD90-APC (BD),anti-cardiac isoform of cTNT (clone 13-11; 1:2000, Thermo Fischer),rabbit anti-human NKX2-5 (1:800, Cell Signaling Technology), goatanti-mouse IgG APC (1:250, BD), goat anti-mouse IgG PE (1:200, JacksonImmunoResearch), donkey anti-rabbit IgG A647 Life Technologies). Forcell-surface markers staining was carried out in PBS containing 5% FCS.For intra-cellular staining cells were fixed for 10 min at 4° C. with 4%PFA and stained in PBS containing 5% FCS and 0.5% saponin (cTNT) or 0.3%TritonX (NKX2-5). Stained cells were analyzed using the LSR II Flowcytometer (BD). For cell sorting the cells were kept in IMDM containing5% FCS and sorted at a concentration of 10⁶ cell/ml using MoFlo (BD) andInflux (BD) sorter. For sorting of PFA-fixed cells for subsequent RNAisolation the staining buffers and PBS for cell collection weresupplemented with 5 mM DTT and 100 U/ml RNaseOUT (Life Technologies).All buffer components were RNAse free and samples were strictly kept onice and sorted within 3 h from PFA-fixation. Data were analyzed usingFlowJo software (Tree Star).

Immunocytochemistry

Cells were fixed with 4% PFA for 10 min at 4° C. and permeabilized using0.3% Triton X, 200 mM Glycerin for 20 min at RT. Samples were blockedfor 30 min at room temperature in blocking buffer (BB): 10% donkeyserum, 2% BSA, 0.1% Triton X. The following primary antibodies wereincubated in BB over night at 4° C.: anti-cardiac isoform of cTNT (clone13-11; 1:100, Thermo Fischer), rabbit anti-human MLC2v (1:100), rabbitanti-human SHOX2 (1:200), (Abcam), rabbit anti-human TBX3 (1:100), goatanti-human Tbx18 (1:50), (Santa Cruz). Respective secondary antibodieswere incubated for 30 min at RT: donkey anti-mouse IgG A647 (1:400),donkey anti-rabbit IgG A467 (1:400), (Life Technologies), goatanti-mouse IgG Cy3 (1:400), donkey anti-rabbit IgG Cy3 (1:800), donkeyanti-goat IgG Cy3 (1:800), (Jackson ImmunoResearch). DAPI was used tocounterstain cell nuclei and slides were mounted using FluorescentMounting Medium (Dako). The Olympus FluoView 1000 Laser ScanningConfocal Microscope and the FV10-software were used for imaging.

For the quantification of DAPI and TBX18 positive cell nuclei the IMAGEJ software plugin ICTN was used. For each condition three random takenpictures (FIG. 3F) of three independent experiments were analyzed.

Quantitative Real-Time PCR

Total RNA was isolated using the RNAqueous-micro Kit (Ambion) followedby a DNase digestion step (Ambion). For RNA isolation from PFA-fixedcells the RecoverAll Kit (Ambion) was used. 500 ng to 1 μg of RNA wasreverse transcribed into cDNA using random hexamers and Oligo(dT)primers with Superscript III Reverse Transcriptase (life technologies).QPCRs were performed on the EP RealPlex MasterCycler (Eppendorf) usingthe QuntiFast SYBR Green PCR Kit (Qiagen) according to themanufacturer's instructions. A 10-fold dilution series of human genomicDNA standards ranging from 25 ng/μl to 2.5 pg/μl was used to evaluatethe efficiency of the PCR and calculate the copy number of each generelative to the house keeping gene TBP as described previously¹³. Primersequences are listed in Table 3. Human fetal heart tissue gestationstage 17 was purchased from Novogenix Laboratories. After dissectingventricular, sinoatrial node and atrioventricular node tissue RNA wasisolated using the Trizol method (Life Technologies) and treated withDNase (Ambion).

Patch Clamp

Action potentials and membrane currents were measured with standardcurrent- and voltage-clamp techniques using axopatch amplifier. For dataacquisition and analysis pCLAMP software (Molecular Devices) was used.Borosilicate glass microelectrodes had tip resistances of 2-5 MΩ whenfilled with pipette solution. Series resistance and cell capacitancewere compensated up to 70%. Spontaneous action potentials, funny current(I_(f)) and inward rectifier potassium currents currents (IK_(r)) wererecorded at 37° C. using the perforated patch method (nystatin)¹⁴ withthe following bath solution (mM): NaCl 124, KCl 5.4, CaCl₂ 1.2, MgCl₂ 1,and glucose 5, Hepes 10 (pH 7.4, adjusted with NaOH). The pipettesolution consisted of (mM): NaCl 5, KCl 10, potassium gluconate 130,MgCl₂ 1, and Hepes 10 (pH 7.4, adjusted with KOH). Sodium currents(I_(Na)) were recorded at 25° C. using the whole cell ruptured patchmethod in calcium free bath solution containing low Na⁺ to reducecurrent amplitude for better voltage control (mM): NaCl 30, TEA-CI 115,CaCl₂ 0.01, MgCl₂ 1, and glucose 5, Hepes 10 (pH 7.4, adjusted withNaOH). The pipette solution consisted of (mM): Cs-aspartate 112, CsCl20, MgCl₂ 1, Mg-ATP 5, Cs-EGTA 10, Hepes 10 (pH 7.4, adjusted withCsOH). The voltage protocols to elicit the individual currents are shownin the respective figures.

Multielectrode Array Experiments

For in-vitro pacing experiments 1×10⁶ VLCM were plated onmatrigel-coated Multielectrode arrays (MEA) and cultured for 5-7 days toform electrical integrated monolayers. Aggregates (3×10⁴ cells) ofSANLCMs or VLCMs (control) labeled with Tetramethylrhodamine methylester (TMRM) were placed at a specific site on these established VLCMmonolayers. Electric signals (Field potentials) were recorded in basicmedia at 37° C. using the Multichannel systems amplifier, heating unitand Cardio-2D software. Greyscale-Maps of electric signal propagationwere generated using the Cardio-2D+ software (multichannelsystems).

Statistics

Data presented as means±standard error of the mean. Statistical analyseswere performed using Student's T-test. Results were considered to besignificant at p<0.05 (*/^(#)) and very significant at p<0.01(**/^(##)).

Results

To generate hESC-derived cardiovascular cells a developmentally stagedprotocol^(6,15) that involves the formation of a primitive streak-likepopulation (days 2-3) expressing T(BRACHYURY) followed by the inductionof cardiac mesoderm characterized by the expression of MESP1 andPDGFRα/KDR (days 3-4) was used. This PDGFRα⁺ KDR⁺ mesoderm gives rise to60-90% cTNT⁺ cardiomyocytes (day 20) (FIG. 1A and FIG. 5A, B). With thisprotocol, expression of transcription factors involved in thedevelopment of the sinoatrial node (SAN)¹⁶ including TBX18, SHOX2 andTBX3 was upregulated, between days 3 and 8 of differentiation,suggesting that pacemaker cells are being specified under theseconditions (FIG. 1B). As studies in both mouse and human indicate thatSAN cells derive from a progenitor that does not express thepan-cardiomyocyte transcription factor NKX2-5¹⁷⁻¹⁹, it was hypothesizedthat it would be possible to distinguish the hPSC-derived pacemakercells from other cardiomyocytes based on expression of NKX2-5. To testthis, the cultures were monitored for the presence of NKX2-5⁺/NKX2-5⁻cardiomyocytes, using the HES3 NKX2-5:GFP reporter line¹⁰ in combinationwith the pan-cardiomyocyte surface marker SIRPA¹³. The firstNKX2-5:GFP⁺SIRPA⁺ cells were generated within 6 days of differentiationand the size of the population increased to represent 60±3% of theculture by day 16 (FIG. 1C). Also, a distinct NKX2-5:GFP⁻SIRPA⁺population was detected by day 16. The cardiomyocyte nature of thesecells was confirmed by cTNT staining which revealed a large (63±8%)NKX2-5:GFP⁺cTNT⁺ population and a small (6±2%) NKX2-5:GFP⁻cTNT⁺population.

To determine if the NKX2-5:GFP⁻ cardiomyocytes represent SAN pacemakercells, the NKX2-5:GFP⁺SIRPA⁺ (NKX2-5⁺) and the NKX2-5:GFP⁻SIRPA⁺(NKX2-5⁻) fractions from day 20 populations were isolated. Themesenchymal marker CD90 was included to deplete non-myocytes from theNKX2-5:GFP⁻ fraction (FIG. 1D and FIG. 5C). Consistent with a pacemakerphenotype, aggregates derived from the NKX2-5⁻ population hadsignificantly faster beating rates (83±4 bpm) than the NKX2-5⁺aggregates (40±3 bpm) (FIG. 1E).

Molecular analyses revealed that the NKX2-5⁺ and NKX2-5⁻ populationsexpressed similar levels of cTNT indicating comparable cardiomyocytecontent (FIG. 1F). NKX2-5⁻ cells expressed significantly less NKX2-5,than the NKX2-5⁺ cells, consistent with the sorting strategy. Theventricular markers MYL2 and IRX4 were expressed at higher levels inNKX2-5⁺ cells than in NKX2-5⁻ cells, indicating that the NKX2-5⁺population contains ventricular-like cardiomyocytes. Genes encodingpacemaker-specific transcription factors including TBX18, TBX3, SHOX2and ISL1 showed the opposite pattern and were express at significantlyhigher levels in NKX2-5⁻ cells (FIG. 1G). Fetal heart tissues includedconfirmed that the SAN markers TBX18, SHOX2 and ISL1 established in themouse heart¹⁶ were also expressed at higher levels in human SAN tissue(F-SAN) compared to ventricular tissue (F-V) and secondary pacemakeratrioventricular node tissue (F-AVN). Accordingly, markers that definethe AVN pacemaker including TBX2 and MSX2^(20,21) were expressed athighest levels in the AVN tissue (FIG. 1H). Expression of thesesAVN-specific genes was not upregulated in the hESC-derived NKX2-5⁻population indicating that it does not contain AVN cells. The pacemakerion channels genes, HCN4, HCN1 and KCNJ3 were expressed at higher levelsin NKX2-5⁻ cells compared to NKX2-5⁺ cells (FIG. 1I), while the sodiumchannel SCN5a showed the opposite pattern. These expression profileswere validated in populations isolated at day 20 by intracellular FACSbased on cTNT and NKX2-5 expression (NKX2-5⁺/cTNT⁺ and NKX2-5/cTNT⁺)(FIG. 5D-H).

Immunostaining confirmed the findings from the molecular studies andshowed that NKX2-5⁺ but not NKX2-5⁻ myocytes expressed the ventricularprotein MLC2V as well as NKX2-5 (FIG. 1J). SHOX2 and TBX3 showed thereverse pattern and were detected at much higher levels in NKX2-5⁻cells. (FIG. 1K and FIG. 6A). When maintained in culture for up to 30days only a small proportion (5±1%) of the NKX2-5⁻ fraction upregulatedexpression of NKX2-5:GFP, indicating that the majority of these cellsrepresent a distinct sub-population of cardiomyocytes rather thanimmature progenitors that have not initiated NKX2-5 expression (FIG. 6B,C).

Although the existing protocol promoted the development of SANLCMs, theefficiency of generating these cells was low (5-9%). Therefore, ahESC-differentiation model was used to gain a better understanding ofSAN lineage development. For this the effect of manipulating signalingpathways was investigated at two distinct stages; mesoderm induction(day 1-3) and cardiovascular specification (day 3-5). For the mesoderminduction step, the concentrations of BMP4 and ACTA was varied as thesesignaling pathways regulate the development of cardiovascular mesoderm⁶.Efficient cardiomyocyte differentiation (70-75%) was achieved with threedifferent combinations of these pathway agonists; standardconcentrations, 10B/6A (10 ng/ml BMP4, 6 ng/ml ACTA) and lowerconcentrations including 5B/4A (5 ng/ml BMP4, 4 ng/ml ACTA) and 3B/2A (3ng/ml BMP4, 2 ng/ml ACTA). (FIG. 7A). None of these combinations led toan increase in the proportion of SANLCMs (7±3% at 10B/6A vs 8±3% at5B/4A and 8±2% at 3B/2A).

Given that the epicardial lineage, a cell type developing from a commonprogenitor with SAN pacemakers is specified from cardiovascular mesodermby BMP4 signaling²², it was next tested if manipulating the BMP4 pathwaywould impact the development of SANLCMs from the three differentmesoderms (FIG. 2A). As reported previously²², blocking BMP signaling(dorsomorphin) or exposure to high levels of BMP4 (10-20 ng/ml) betweenday 3-5 of differentiation resulted in a significant reduction of totalcardiomyocytes (FIG. 2B-D and FIG. 7B). Importantly, lower levels ofBMP4 (1.25-5 ng/ml) led to a notable increase in the proportion ofSANLCMs up to 35±1%. Under these conditions, the NKX-2-5:GFP⁻ SANLCMscould be easily resolved as a distinct population, based on cTNT/SIRPAexpression (FIG. 2E). The effect was greatest in the mesoderm inducedwith 3B/2A (35±1% vs 15±2% at 10B/6A vs 21±1% at 5B/4A), suggesting thatSANLCMs derive from a distinct subpopulation of mesoderm that is inducedwith low concentration of BMP4 and ACTA. The increase in the proportionof SANLCMs was most pronounced if BMP4 was added between days 3 and 4 ofdifferentiation (FIG. 2F). Addition of BMP4 for longer periods of timedid not increase the proportion of SANLCMs, whereas activation of thepathway earlier, (days 2-3, 2-4) or later (days 4-5, 4-6) resulted in areduction in the frequency of SANLCMs. These observations highlight thedynamic nature of lineage specification in hPSC differentiations and theimportance of manipulating signaling pathways at the appropriatedevelopmental stage.

Given that the SAN derives from a TBX18⁺ progenitor, TBX18 expressionduring BMP4-induced SANLCM specification was monitored. Consistent withthe initial analyses (FIG. 1B), TBX18 expression was upregulated betweendays 4 and 6 of differentiation and treatment with BMP4 significantlyup-regulated TBX18 (FIG. 2G). Immunostaining confirmed the expressionstudies and showed that the BMP4-induced population containedsignificantly more TBX18⁺ cells than the control population (FIG. 2H,I). Notably the number of TBX18⁺ cells at day 6 (38±2%) correlated withthe number of SANLCMs at day 20 (35±1%), a finding supporting theinterpretation that SANLCMs derived from a TBX18⁺ progenitor.

Retinoic acid (RA) signaling is known to play a pivotal role in thegeneration of atrial cardiomyocytes that derive from progenitorspositioned in the posterior region of the heart tube^(23,24). As the SANalso develops from the posterior heart tube, it was next asked if RAsignaling impacts the development of SANLCMs. Addition of RA (500 nMe.g. about 150 ng/mL) at day 2 blocked the generation of cardiomyocytes(FIG. 3A). When added at later times (days 3 to 12), RA had no effect onthe generation of total cardiomyocytes and SANLCM frequency. Although RAdid not affect the efficiency of SANLCM development, qRT-PCR analyses ofthe isolated NKX2-5⁻SIRPA⁺ fraction revealed a significant upregulationof the posterior cardiomyocyte marker TBX5 and the SAN marker SHOX2,when RA was added to the cultures between days 3 to 5 (FIG. 3B).

Importantly, addition of RA (day 3) did not impact the BMP4-inducedincrease in SANLCM specification (FIG. 3c and FIG. 8A) nor did it alterthe levels of TBX18 expression (FIG. 3D). RA treatment alone and incombination with BMP4 increased the expression of TBX5, SHOX2 and ISL1and further decreased the expression of MYL2 (FIG. 3E). Combined RA/BMP4signaling significantly increased the expression of the pacemaker ionchannel genes HCN4 and HCN1 but not the AVN genes TBX2 and MSX2 (FIG.3F). The improved RA/BMP-induced pacemaker expression profile wasassociated with a significant increase in the beating rate of the cells(138±7 bpm) (FIG. 3G). These rates are within the range of the humanfetal heartbeat which is at 120-160 bpm²⁵.

Taken together, these findings support a model in which the SAN lineageis specified from an appropriately induced mesoderm population (3B/2A)by BMP signaling through a TBX18⁺ progenitor. RA signaling by contrastdoes not affect the efficiency of SAN lineage specification but ratherenhances the pacemaker phenotype in SAN progenitors (FIG. 3H). Usingthis strategy SANLCM could be generated from the H7 hESC line and theMSC-iPS1 hiPSC line (FIG. 8B-G).

Electrophysiological analyses revealed that SANLCM had significantlyfaster spontaneous action potential rates compared to VLCMs (FIG. 4A,B). Ninety percent of SANLCMs showed action potentials with typicalpacemaker characteristics including slow maximum upstroke velocity (<30V/s), small action potential amplitude and short action potentialduration (FIG. 4C, Table 1). Additionally, SANLCMs containedsignificantly more pacemaker funny current (I_(f)) and less sodiumcurrent (I_(Na)), than the VLCMs (FIG. 4D, E and FIG. 9A-D). Inaddition, less inward rectifier potassium current (I_(K1)) was observedin SANLCMS, consistent with the hyperpolarized diastolic membranepotential of pacemaker cells (FIG. 9E, F).

To determine if SANLCMs could function as pacemakers, their ability tocontrol the beating rate of VLCMs was tested. For these analyses, VLCMswere isolated by FACS and plated on a multi-electrode array (MEA) toform electrically integrated monolayers. Aggregates of SANLCMs or VLCMs(control) labeled with Tetramethylrhodamine methyl ester (TMRM) wereplaced at a specific site on these established VLCM monolayers. Thecombined aggregate/monolayer populations were cultured for a week toallow electrical integration (FIG. 4F). Analysis of electric signalpropagation showed that, in the absence of any aggregates, theinitiation site of electric activity in the monolayers changed randomly(FIG. 4G-I, FIG. 10A-C). In contrast, electric activity was stablyinitiated by the SANLCM aggregate (electrode 65) and propagated throughthe adjacent monolayer (FIG. 4J-L). As a result of this pacing activity,the beating frequency of the monolayer increased from 63.1±2.5 to112.5±18.5 bpm (Table 2). In seventy-five percent of the experimentsSANLCM were able to pace the VLCM monolayer for the 14-day duration ofthe experiment. Aggregates of VLCM did not show this pacing capacity, asinitiation of electric activity remained random in these cultures (FIG.10D-F).

During embryonic heart development FGF signaling secreted by the neuralectoderm is involved in the specification of NKX2-5⁺ cardiacprogenitors²⁶. We reasoned that inhibition of FGF signaling couldrepress development of the NKX2-5⁺ progenitors favoring the generationof the NKX2-5⁻ SANLCMs. We therefore applied the FGF receptor inhibitorPD 173074 (Tocris) to our pacemaker differentiation conditions (BMP+RA)and found that inhibition of FGF signaling from day 4-6 results in anincrease of NKX2-5⁻cTNT⁺ SANLCM from 33±1% to 48±3% in day 20 cultures.In contrast activation of FGF signaling using bFGF decreased the SANLCMpopulation (12±1%). Importantly, application of the FGF inhibitor in aconcentration ranging from 240-960 nM blocked the development of NKX2-5⁺cardiomyocytes (5±1% vs 35±5% at endogenous (e) bFGF levels e.g. no FGFadded to the media). The block in development of NKX2-5⁺ cardiomyocyteswas most efficient when the FGF inhibitor was applied between day3-4 buthad no effect when it was applied from day 5 onwards. This suggests thatthe NKX2-5⁺ progenitor is specified around day 3-4, which correlateswith the first detection of NK2-5:GFP⁺ cells at day 6 ofdifferentiation. (FIG. 12A-D).

Since the FGF inhibitor treated cultures only contain a small number ofNKX2-5⁺ cardiomyocytes it allows to isolate relatively pure populationsof SANLCMs by selecting for SIRPA⁺ and CD90⁻ cardiomyocytes (76%post-sort purity for NKX2-5⁻cTNT⁺ SANLCMs) (FIG. 12E). Using thisapproach it should be possible to obtain highly enriched populations ofSANLCMs from any human pluripotent stem cell line, independent of theNKX2-5:GFP transgene expression. As a proof of principle we applied ourprotocol to the HES-2 embryonic stem cell line. We specified the cardiacmesoderm using 2.5 ng/ml BMP and 0.5 μM RA in the presence of the FGFinhibitor. To analyze the cultures for the SANLCM proportion, at day 20intracellular staining for NKX2-5 protein and cTNT followed by flowcytometric analysis was applied. For the HES2 line treatment with 240 nMFGFi from day3-5 obtained best results and generated cultures with up to36% NKX2-5⁻cTNT⁺ SANLCMs and only a small proportion of NKX2-5⁺ myocytes(5% vs 43% at endogenous bFGF levels) (FIG. 12F).

Human pacemaker cells have previously been isolated from hPSCs-derivedcardiomyocyte populations using a cGata6 reporter²⁷. Although thephenotype of these pacemaker cells was not specified they most likelyresemble the secondary AVN pacemaker as the cGata6 reporter specificallymarks the AVN in the mouse heart²⁸. Here it is shown for the first timethat pacemaker cells resembling the SAN can be isolated fromhPSCs-derived cardiomyocyte populations using a NKX2-5:GFP reporter. Twonovel signaling pathways BMP4 and RA that control SAN lineagespecification were identified. The benefit of understanding pacemakerdevelopment is twofold because it also enables the elimination ofcontaminating pacemakers from hPSCs-derived ventricular populationswhere they could cause life-threatening arrhythmias upon transplantationinto the heart. Accordingly, SANLCM present an attractive source for thegeneration of a biological pacemaker. Current approaches to a biologicalpacemaker include overexpression of pacemaker ion-channels andreprogramming of existing working cardiomyocytes^(29,30). It issuggested that hPSC-derived SANLCM represent a genuine biologicalpacemaker as they are generated by recapitulation of human development.

Example 2

Methods for In-vivo Transplantation and Ex Vivo SimultaneousECG-recordings and Optical Mapping

For the assessment of in vivo pacemaker capacity of SANLCM and VLCM, 8adult Fischer-344 (200-300 g) rats were used. Animals were anesthetized(87 mg/kg Ketamine, 13 mg/kg Xylazine), intubated andmechanically-ventilated (100% O₂, 1 ml/kg volume). Following a leftthoracotomy, 1-2×10⁶ SANLCM or VLCM (aggregated in low cluster 96 wellplates at 80,000 cells/well) were injected into the left-ventricularanterior wall near the apex using a 28G needle. To prevent immunerejection of the human cell-grafts, animals were treated withcyclosporine A (13 mg/kg/day) and methylprednisolone (2 mg/kg/day).

For the optical mapping studies, at 14 days post-transplantation, heartswere harvested, transferred to a custom-built optical mapping chamber(allowing simultaneous electrocardiographic recordings and fluorescentvoltage imaging) and retrogradely perfused using a Langendorff apparatuswith oxygenized Tyrod's solution.

A unipolar electromyogram was recorded using a digital ECG dataacquisition system (Biopac systems). A high-speed CCD-based opticalmapping technique (Scimedia) was used to study the electrical activationpatterns of the rat heart. To this end, the voltage-sensitive dyeDi-4-ANBDQBS (40 μL, 29.61 mMol/L) was added to the perfusate forloading prior to voltage mapping. Optical mapping was performed byillumination of the hearts with 1,000 watt quartz tungsten halogen lamp(Newport Corporation, USA), equipped with an electronic shutter and withan excitation filter of 660±10 nm (Chroma Technology Corp). Emission wasmeasured using long-pass filter >715 nm (Edmund Optics) adjacent to theCCD camera.

A custom-based computer software (OMproCCD) was utilized for dataanalysis of the optical mapping signals. The data was viewed either asdynamic displays (movies) showing the propagation of the electricactivation wavefronts or as activation maps. Activation (isochronal)maps were constructed by measuring the timing of electrical activationat each imaged-pixel (timing of the maximum dF/dt). The hearts weremapped at baseline (sinus-rhythm) and following application ofMethacholine (1 μM, 0.1 ml) and Lidocaine (0.005% 0.1 ml) aiming toinduce complete AV block and suppress the junctional escape rhythm.

The ability of SANLCMs to function as pacemaker in-vivo was tested byengrafting them as cell-aggregates into the apex of the rat heart. Toevaluate the effect of cell transplantation 14 days later, opticalmapping was performed using the isolated Langendorff-perfused heartmodel. The heart-rate of the rat is ˜300 bpm, much faster thanbeating-rate of the human heart. A pharmacological approach wastherefore developed (application of 0.1 ml Metacholine (1 μM)+Lidocaine(0.005%)) to induce transient complete atrioventricular (AV) block andsuppress ventricular escape rhythm, in order to reveal potentialpacemaker activity of the transplant. Using this approach three of threehearts that were injected with control VLCM demonstrated complete AVblock with a junctional escape rhythm that slowed down to 76±4 bpm (FIG.11A). Optical mapping confirmed that this endogenous slow escape rhythmwas initiated at the septum (away for the area of cell transplantation)and then propagated to activate the rest of the ventricle (FIG. 11B). Incontrast three out of three hearts that received the SANLCM transplant,displayed a significantly faster ventricular ectopic rhythm afterinduction of AV block (145±8 bpm). Optical mapping revealed that thisnew ectopic rhythm was initiated from the apex of the heart matching thetransplantation site (FIG. 11C,D). The presence of the transplant andits ventricular/pacemaker identity was confirmed by immunostaining forhuman specific cTNT and MLC2v (FIG. 11E,F) Taken together theseexperiments provide the proof of principle that purified SANLCMs can actas biological pacemaker both in-vitro and in-vivo while VLCM have nopacemaker capacity.

TABLE 1 Electrophysiological characteristics of VLCM and SANLCM N Rate(bpm) dv/dt_(max) (V/s) DMP (mV) APA (mV) APD50 (ms) APD90 (ms) VLCMs 2273.1 ± 6.3  115.9 ± 13.9 −64.1 ± 1.7  121.6 ± 5.3   130.4 ± 13.8   194.3± 14.9  SANLCMs 19 133.1 ± 4.5**  11.9 ± 3.1** −55.8 ± 2.6** 88.3 ±2.9** 65.4 ± 12.3** 147.2 ± 13.1* APA, action potential amplitude;APD50, action potential duration at 50% of repolarization; APD90, actionpotential duration at 90% of repolarization; DMP, diastolic membranepotential; dv/dt_(max), maximum action potential upstroke velocity; N,cell number; *P < 0.05, **P < 0.01 vs VLCM

TABLE 2 Efficiency of VLCM and SANLCM aggregates to pace VLCM MonolayersMonolayer Electrical Monolayer Rate before Aggregate integration PacingPacing Rate after Aggregates N (bpm) Rate (bpm) (n) (n) (%) (bpm) VLCMs6 62.6 ± 2.1 53.5 ± 6.6  6 0  0% 54.5 ± 10.7  SANLCMs 9 63.1 ± 2.5 142.2± 7.4** 8 6 75% 112.5 ± 18.5*¹ ¹Beating rates were only accounted for insuccessfully paced cultures. Monolayer Rate before, beating rate ofmonolayer before placing the respective aggregate; Monolayer Rate after,beating rate of monolayer after placing the respective aggregate; N,cell number; *P < 0.05, **P < 0.01 vs VLCM Aggregates

TABLE 3 Primer Sequences Gene Forward 5′-3′ (SEQ ID NO) Reverse 5′-3′(SEQ ID NO) BRACHYURY TGTCCCAGGTGGCTTACAGATGA GGTGTGCCAAAGTTGCCAATACA(T) (1) (2) cTNT TTCACCAAAGATCTGCTCCTCGCT TTATTACTGGTGTGGAGTGGGTGTGG (3)(4) HCN1 GCAGGCAATCGCTCCCATCAATTA TGTGTACACCGGTGGAGATTGTGT (5) (6) HCN4TCTTCCTCATTGTGGAGACACGCA TGAGGATCTTCGTGAAGCGGACAA (7) (8) IRX4TTGGACTCCTGGGAACATGGACAA ATGCTTCAGGGTATCTGGCCTCTT (9) (10) ISL1GAAGGTGGAGCTGCATTGGTT TAAACCAGCTACAGGACAGGCC (11) (12) KCNJ3TCATCAAGATGTCCCAGCCCAAGA CACCCGGAACATAAGCGTGAGTTT (13) (14) MESP1AGCCCAAGTGACAAGGGACA AAGGAACCACTTCGAAGGTGC (15) (16) MSX2GCGCAAGTTCCGTCAGAAACAGTA TTTGACCTGGGTCTCTGTGAGGTT (17) (18) MYL2TGTCCCTACCTTGTCTGTTAG ATTGGAACATGGCCTCTGGAT (19) (20) NKX2-5TTTGCATTCACTCCTGCGGAGACC ACTCATTGCACGCTGCATAATCG (21) (22) SCN5ATGCTGCTCTTCCTCGTCATGTTCA TGTTGGCGAAGGTCTGGAAGTTGA (23) (24) SHOX2ATCGCAAAGAGGATGCGAAAGGGA TTCCAGGGTGAAATTGGTCCGACT (25) (26) TBPTGAGTTGCTCATACCGTGCTG CCCTCAAACCAACTTGTCAACAG (27) (28) TBX2AACGCATGTACATCCACCCAGACA TTGTTGGTCAGCTTCAGCTTGTGG (29) (30) TBX3TTGAAGACCATGGAGCCCGAAGAA CCCGCTTGTGAAACTGATCCCAAA (31) (32) TBX5ACAAAGTGAAGGTGACGGGCCTTA ATCTGTGATCGTCGGCAGGTACAA (33) (34) TBX18TTAACCTTGTCCGTCTGCCTGAGT GTAATGGGCTTTGGCCTTTGCACT (35) (36)

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

-   1 Boyett, M. R., Honjo, H. & Kodama, I. The sinoatrial node, a    heterogeneous pacemaker structure. Cardiovascular research 47,    658-687 (2000).-   2 Monfredi, O., Dobrzynski, H., Mondal, T., Boyett, M. R. &    Morris, G. M. The anatomy and physiology of the sinoatrial node—a    contemporary review. Pacing and clinical electrophysiology: PACE 33,    1392-1406, doi:10.1111/j.1540-8159.2010.02838.x (2010).-   3 Nof, E., Glikson, M. & Antzelevitch, C. Genetics and Sinus Node    Dysfunction. Journal of atrial fibrillation 1, 328-336 (2009).-   4 Lau, D. H., Roberts-Thomson, K. C. & Sanders, P. Sinus node    revisited. Current opinion in cardiology 26, 55-59,    doi:10.1097/HCO.0b013e32834138f4 (2011).-   5 Laflamme, M. A. et al. Cardiomyocytes derived from human embryonic    stem cells in pro-survival factors enhance function of infarcted rat    hearts. Nature biotechnology 25, 1015-1024, doi:10.1038/nbt1327    (2007).-   6 Kattman, S. J. et al. Stage-specific optimization of activin/nodal    and BMP signaling promotes cardiac differentiation of mouse and    human pluripotent stem cell lines. Cell stem cell 8, 228-240,    doi:10.1016/j.stem.2010.12.008 (2011).-   7 Burridge, P. W. et al. Chemically defined generation of human    cardiomyocytes. Nature methods 11, 855-860, doi:10.1038/nmeth.2999    (2014).-   8 He, J. Q., Ma, Y., Lee, Y., Thomson, J. A. & Kamp, T. J. Human    embryonic stem cells develop into multiple types of cardiac    myocytes: action potential characterization. Circulation research    93, 32-39, doi:10.1161/01.RES.0000080317.92718.99 (2003).-   9 Ma, J. et al. High purity human-induced pluripotent stem    cell-derived cardiomyocytes: electrophysiological properties of    action potentials and ionic currents. American journal of    physiology. Heart and circulatory physiology 301, H2006-2017,    doi:10.1152/ajpheart.00694.2011 (2011).-   10 Elliott, D. A. et al. NKX2-5(eGFP/w) hESCs for isolation of human    cardiac progenitors and cardiomyocytes. Nature methods 8, 1037-1040,    doi:10.1038/nmeth.1740 (2011).-   11 Park, I. H. et al. Reprogramming of human somatic cells to    pluripotency with defined factors. Nature 451, 141-146,    doi:10.1038/nature06534 (2008).-   12 Kennedy, M., D'Souza, S. L., Lynch-Kattman, M., Schwantz, S. &    Keller, G. Development of the hemangioblast defines the onset of    hematopoiesis in human ES cell differentiation cultures. Blood 109,    2679-2687, doi:10.1182/blood-2006-09-047704 (2007).-   13 Dubois, N. C. et al. SIRPA is a specific cell-surface marker for    isolating cardiomyocytes derived from human pluripotent stem cells.    Nature biotechnology 29, 1011-1018, doi:10.1038/nbt.2005 (2011).-   14 Okada, Y. & SpringerLink (Online service). in Springer Protocols    Handbooks, (Springer Japan, Tokyo, 2012).-   15 Yang, L. et al. Human cardiovascular progenitor cells develop    from a KDR+ embryonic-stem-cell-derived population. Nature 453,    524-528, doi:10.1038/nature06894 (2008).-   16 Christoffels, V. M., Smits, G. J., Kispert, A. & Moorman, A. F.    Development of the pacemaker tissues of the heart. Circulation    research 106, 240-254, doi:10.1161/CIRCRESAHA.109.205419 (2010).-   17 Mommersteeg, M. T. et al. The sinus venosus progenitors separate    and diversify from the first and second heart fields early in    development. Cardiovascular research 87, 92-101,    doi:10.1093/cvr/cvq033 (2010).-   18 Sizarov, A. et al. Molecular analysis of patterning of conduction    tissues in the developing human heart. Circulation. Arrhythmia and    electrophysiology 4, 532-542, doi:10.1161/CIRCEP.111.963421 (2011).-   19 Christoffels, V. M. et al. Formation of the venous pole of the    heart from an NKX2-5-negative precursor population requires Tbx18.    Circulation research 98, 1555-1563,    doi:10.1161/01.RES.0000227571.84189.65 (2006).-   20 Christoffels, V. M. & Moorman, A. F. Development of the cardiac    conduction system: why are some regions of the heart more    arrhythmogenic than others? Circulation. Arrhythmia and    electrophysiology 2, 195-207, doi:10.1161/CIRCEP.108.829341 (2009).-   21 Horsthuis, T. et al. Gene expression profiling of the forming    atrioventricular node using a novel tbx3-based node-specific    transgenic reporter. Circulation research 105, 61-69,    doi:10.1161/CIRCRESAHA.108.192443 (2009).-   22 Witty, A. D. et al. Generation of the epicardial lineage from    human pluripotent stem cells. Nature biotechnology,    doi:10.1038/nbt.3002 (2014).-   23 Xavier-Neto, J. et al. A retinoic acid-inducible transgenic    marker of sino-atrial development in the mouse heart. Development    126, 2677-2687 (1999).-   24 Rosenthal, N. & Xavier-Neto, J. From the bottom of the heart:    anteroposterior decisions in cardiac muscle differentiation. Current    opinion in cell biology 12, 742-746 (2000).-   25 Pildner von Steinburg, S. et al. What is the “normal” fetal heart    rate? PeerJ 1, e82, doi:10.7717/peerj.82 (2013).-   26 Keren-Politansky, A., Keren, A. & Bengal, E. Neural    ectoderm-secreted FGF initiates the expression of NKX2.5 in cardiac    progenitors via a p38 MAPK/CREB pathway. Developmental biology 335,    374-384, doi:10.1016/j.ydbio.2009.09.012 (2009).-   27 Zhu, W. Z. et al. Neuregulin/ErbB signaling regulates cardiac    subtype specification in differentiating human embryonic stem cells.    Circulation research 107, 776-786, doi:10.1161/CIRCRESAHA.110.223917    (2010).-   28 Davis, D. L. et al. A GATA-6 gene heart-region-specific enhancer    provides a novel means to mark and probe a discrete component of the    mouse cardiac conduction system. Mechanisms of development 108,    105-119 (2001).-   29 Li, R. A. Gene- and cell-based bio-artificial pacemaker: what    basic and translational lessons have we learned? Gene therapy 19,    588-595, doi:10.1038/gt.2012.33 (2012).-   30 Hu, Y. F., Dawkins, J. F., Cho, H. C., Marban, E. & Cingolani, E.    Biological pacemaker created by minimally invasive somatic    reprogramming in pigs with complete heart block. Science    translational medicine 6, 245ra294, doi:10.1126/scitranslmed.3008681    (2014).

The invention claimed is:
 1. A method of producing a population ofcardiomyocytes, the method comprising: (a) incubating cardiovascularmesoderm cells in a cardiac induction medium comprising a WNT inhibitor,VEGF, or both for a period of time to generate cardiovascular progenitorcells that express NKX2-5; and (b) incubating the cardiovascularprogenitor cells in a basic medium comprising VEGF for a period of timeto generated a population of cardiomyocytes that are enriched for NKX2-5^(pos), cTNT^(pos) cells.
 2. The method of claim 1, wherein thecardiovascular mesoderm cells are generated from pluripotent stem cells(PSCs) according to the method: (a) incubating the PSCs in an embryoidbody medium comprising a BMP component for a period of time to generateembryoid bodies; and (b) incubating the embryoid bodies in a mesoderminduction medium comprising a BMP component and an activin component fora period of time to generate cardiovascular mesoderm cells.
 3. Themethod of claim 2, wherein the PSCs are human PSCs (hPSCs).
 4. Themethod of claim 3, wherein the hPSCs are induced pluripotent stem cells(iPSCs) or human embryonic stem cells (hESCs).
 5. The method of claim 3,wherein the hPSCs are hPSCs comprising a NKX2-5 reporter construct. 6.The method of claim 5, further comprising isolating a population ofventricular-like cardiomyocytes (VLCM) from the population ofcardiomyocytes comprising selecting NKX2-5 positive cardiotnyocytesaccording to NKX2-5 reporter construct expression.
 7. The method ofclaim 5, wherein the NKX2-5 reporter construct is a fluorescent NKX2-5reporter construct.
 8. The method of claim 1, wherein the cardiacinduction medium comprises a WNT inhibitor.
 9. The method of claim 8,wherein the cardiac induction medium further comprises VEGF.
 10. Themethod of claim 9, wherein the WNT inhibitor is IWP2.
 11. The method ofclaim 10, wherein the cardiac induction medium further comprises atleast one of a FGF component and an activin/nodal inhibitor.
 12. Themethod of claim 10, wherein the cardiac induction medium furthercomprises SR-431542.
 13. The method of claim 10, further comprising thestep of isolating the population of cardiomyocytes using acardiomyocyte-specific surface marker or markers.
 14. The method ofclaim 13 wherein the step of isolating the population of cardiomyocytesuses the markers signal-regulatory protein alpha (SIRPA) and thymocytedifferentiation antigen 1 (THY-1/CD90).
 15. The method of claim 2,wherein the embryoid body medium BMP component is BMP4 and the mesoderminduction medium BMP component is BMP4.
 16. The method of claim 15,wherein the embryoid body medium further comprises a Rho-associatedprotein kinase (ROCK) inhibitor, the mesoderm induction medium activincomponent is Activin A, and the mesoderm induction medium furthercomprises bFGF.
 17. The method of claim 12, wherein the cardiovascularmesoderm cells are generated from pluripotent stem cells (PSCs)according to the method: (a) incubating the PSCs in an embryoid bodymedium comprising a BMP component for a period of time to generateembryoid bodies; and (b) incubating the embryoid bodies in a mesoderminduction medium comprising a BMP component and an activin component fora period of time to generate cardiovascular mesoderm cells.
 18. Themethod of claim 17, wherein the embryoid body medium BMP component isBMP4 and the mesoderm induction medium BMP component is BMP4.
 19. Themethod of claim 18, wherein the embryoid body medium further comprises aRho-associated protein kinase (ROCK) inhibitor, the mesoderm inductionmedium activin component is Activin A, and the mesoderm induction mediumfurther comprises bFGF.
 20. The method of claim 19, wherein the PSCs arehuman PSCs (hPSCs).
 21. The method of claim 20, wherein the hPSCs arehuman induced pluripotent stem cells (hiPSCs) or human embryonic stemcells (hESCs).
 22. The method of claim 7, wherein the fluorescent NKX2-5reporter construct is a NKX2-5:GFP reporter construct.